SEARCH

SEARCH BY CITATION

Keywords:

  • proteomics;
  • intersexual differences;
  • protein expression;
  • hippocampus

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

It is widely accepted that intersexual differences occur in cognitive domains, e.g., in spatial learning and memory. The hippocampus plays important roles in the consolidation of information from short-term memory to long-term memory and spatial navigation. However, it still remains unknown whether the hippocampal proteomic profiling differs between males and females. In this study, we investigated the intersexual differences in protein expression of hippocampi using the two-dimensional electrophoresis analysis. In all, 33 differentially expressed proteins were characterized by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and validated by Western-blotting analysis. In line with Western-blotting validation, the proteomic identification revealed the overexpression of glial fibrillary acidic protein in female rats' hippocampi, and the overexpression of both creatine kinase B-type and DRP-2 in male rats' hippocampi. The intersexual differences in hippocampal proteomic profiling are probably closely related to those in spatial learning and memory abilities. Anat Rec, 296:462–469, 2013. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Males and females may differ in many aspects of brain activities and behavior, including facial expression, pain perception, navigation, neurotransmitter levels, stress hormone action on the brain and disease states (Cahill, 2006). In both humans and other animals, intersexual differences occur in various perceptual, memory and neural function, motor and cognitive domains (Clements et al., 2006; Hartshorne and Ullman, 2006). These differences may be attributed to various genetic, hormonal and/or environmental factors, but reflect no overall superior advantage to either sex (Zeenat, 2010). Intersexual differences occur in all brain lobes, including many “cognitive” regions such as the hippocampus, amygdala and neocortex (Juraska, 1991).

The hippocampus plays important roles in the consolidation of information from short-term memory to long-term memory and spatial navigation. Males' and females' hippocampi differ significantly in their anatomical structure, their neuroanatomic make-up and their reactivity to stressful situations (Madeira and Lieberman, 1995). Imaging studies showed that the hippocampus is larger in women than in men when adjusted for total brain size (Goldstein et al., 2001). Males' hippocampi respond to both spaced and continuous tetanic stimulation and display long-term potentiation (LTP), while females' hippocampi respond only to continuous stimulation (Yang et al., 2004). Two sexes are known to behave differently in learning and memory tasks (Andreano and Cahill 2009). Previous comparative studies indicated an overall advantage for females on tests of verbal abilities, and for males on tests of quantitative and visual-spatial abilities (Philips and Cupchik, 2003). However, there are exceptions. For example, Geiger and Robert (2005) reported that males have better verbal and spatial memory than females.

It is widely accepted that at least two stages occur in the learning process: short-term memory for recent events (i.e., working memory) and long-term acquired memory for past events (i.e., reference memory) (Gurzu et al., 2008). In short-term memory, information is temporarily stored on the basis of changes in preexisting connections due to covalent modification of preexisting proteins, while in long-term memory, the information is stored more permanently through the growth of new connections as a result of transcription and translation of certain genes, a process called “consolidation” (Touzani et al., 2007). The consolidation of memory implies the involvement of de novo protein syntheses and modulation of gene expression (Goelet et al., 1986). Previous studies have proved that protein syntheses and expression are a prerequisite for the formation of long-term memory in a variety of species and training paradigms (Martin et al., 2000; Naghdi et al., 2003). It was reported that tetanus-induced potentiation of the synaptic response in CA1 and dentate gyrus was relatively short-lived in animals that were injected with protein synthesis inhibitors (Krug et al., 1984; Otani et al., 1989; Mullany and Lynch, 1997; Stanton and Sarvey, 1984, Zheng et al., 2009).

However, it still remains unknown whether the hippocampal proteomic profiling differs between males and females. In this study, we aimed to: (1) evaluate the magnitude of intersexual differences in spatial learning and working memory; (2) investigate the differences in protein expression of hippocampi between male and female rats; and (3) characterize and validate differentially expressed proteins.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Animals

In this study, young adult Sprague Dawley (SD) rats (8–10 weeks; 200–250 g) were used as study animals. In all, eight males and eight females were randomly selected from eight different litters, with one male and one female being from each litter. All experimental rats were purchased from the Medical Experimental Animal Center of Shaanxi Province (China). All animals were housed four per cage, maintained at room temperature (23±2 °C) under standard 12:12 h light–dark cycles. Standard rat chow and water were available ad libitum. Although female rats' performance in the Morris water maze (MWM) may fluctuate across the oestrous cycle (Warren and Juraska, 1997), such an effect was not evaluated in this study since we meant to treat the sample as a heterogeneous population. The female rats were tested randomly throughout their cycles. The Institutional Animal Care Committee of Xi'an Jiaotong University approved the experimental protocol.

MWM

The MWM is designed to test spatial learning and reference memory by observing and recording Escape Latency. The MWM consisted of a circular pool (diameter 1.5 m) located in the center of the testing room. The water maze was surrounded by a number of fixed clues. The pool was filled to a depth of 45 cm with water at 23±1 °C. For the hidden platform task, an escape platform (diameter 12 cm) was located at a fixed position in the southwest quadrant of the maze, and was hidden 2 cm below the surface of the water. Rats received four trials a day over six consecutive days (one training day and five testing day). On the first day during the training task, rats were trained for four trials, starting at four different positions in a clockwise order. A maximum of 120 s was allowed before the rats were assisted to the platform. After climbing onto the hidden platform, rats remained on the platform for 15 s. Then, the platform was removed from the water maze. After the training day, there are five consecutive testing day, during the testing phase, a score of 120 s was recorded for unsuccessful attempts at locating the hidden platform. On the seventh day following the last day of hidden platform testing, platform was removed from the water maze. Spatial activity was expressed by the times of rats crossing the former hidden platform location. More crossings indicated superior spatial memory. The movement of the rats in the task was assessed using a video camera mounted above the maze and interfaced with a computerized tracking system (Water 2020, HVS Image, UK). The computerized tracking system produced a digitized recording of each individual swim trial, which was subsequently used for analyzing performance in the task.

Hippocampal Dissection and Sample Preparation

Eight hours after the probe trials in the MWM, rats were killed and hippocampi were dissected. Tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C until the subsequent use. Great efforts were made to minimize animal suffering and the number of animals used. Within each group, four rats were used for the two-dimensional electrophoresis (2DE) analysis and the other four for the Western-blotting analysis.

The sample preparation followed the protocol of Samara et al. (2011). The frozen hippocampi were washed three times with chilled phosphate-buffered saline (PBS), pulverized in liquid nitrogen and homogenized. Each sample was prepared by mixing four rats' hippocampi (contralateral hippocampi) within the same group. The protein content in the supernatant was determined by the Bradford method.

2DE

2DE was performed as described by Samara et al. (2011). Prior to the first dimension of 2DE, 1 mg of protein sample was diluted to 350 μL with rehydration buffer, and supplemented with 0.5% IPG buffer (pH 3–10 NL, GE Healthcare, Sweden). The isoelectric focusing (IEF) was conducted on an Ettan IPGphor 3 IEF system (GE Healthcare, Sweden) at 20 °C with a current limit of 50 μA per strip. The IPG strips were 18 cm nonlinear pH3–10 (GE Healthcare, Sweden). For each experimental condition, six gels (three for female and three for male mix repeats) were run in parallel under identical 2D electrophoresis conditions. The protein spots were visualized in gel by Coomassie brilliant blue staining upon the completion of electrophoreses. Stained 2D gels were captured by transmission scan (LabScan, Amersham). Target gels were analyzed with the program ImageMaster v5.0 (GE Healthcare, Sweden).

The gels were scanned by Image Scanner III (GE Healthcare, Sweden) at 400 DPI resolutions, and then analyzed by the program ImageMaster 2D Platinum v6.0 (GE Healthcare, Sweden). The intensity of each spot was first processed by background subtraction, and then normalized between gels as a proportion of the total protein intensity from the entire gel. Only the well-separated spots were considered for quantification. Only those with different expression levels (fold ratio male/female>2 or<0.5; P<0.05) between males' and females' hippocampi were further examined for statistical significant differentiation in expression levels using the Student's t-test.

Trypsin Digestion and Protein Identification by MALD-TOF MS

Protein spots of interest were manually excised from the 2D gels, and the in-gel digestion was performed as previously described (Samara et al., 2011). Following that, we spotted 1 μL of a mixture containing 10 μL 0.3 g/L a-cyano-4-hydroxycinnamic acid in 2:1 ethanol/acetone (v/v) and 1 μL of the digested peptides fraction onto the MALDI AnchorChip™ (Bruker Daltonics, Germany). The identification of spots was performed by MALDI-TOF/TOF MS (Bruker Daltonics, Germany). Mass spectra were then acquired for peptide mass fingerprinting. The generated peptide sequence tags were analyzed with the program MASCOT v1.9 (Matrix Science; http://www.matrixscience.co.uk).

Western Blotting Validation

Western blotting was performed to confirm the expression of creatine kinase B-type (CKB), dihydropyrimidinase-related protein 2 (DRP-2), and glial fibrillary acidic protein (GFAP) in the hippocampi of male and female rats. The analyzed samples were derived from the other four separate individuals within each group. The antibodies employed for Western blotting validation experiments were as follows (product numbers and the dilutions of primary antibodies in parentheses): CKB antibody (Santa Cruz Biotechnology; sc-15157; 1:200), DRP-2 antibody (Bioworld Technology; BS3005; 1:500), GFAP antibody (Santa Cruz Biotechnology; sc-32955; 1:100), and β-actin (C4) Antibody (Santa Cruz Biotechnology; sc-47778; 1:100).

A total of 8 μg of each sample was loaded onto 12% Excel Gel SDS homogeneous gels (GE Healthcare, UK). Proteins separated in the gel were transferred onto PVDF membranes (Millipore, Ireland). Membranes were blocked with 1 × PBS containing 2.5 mg/mL BSA and 0.25% (v/v) Tween 20, followed subsequently by the incubation with the primary antibody and by the dilution in PBS at concentrations according to the antibody instruction. Then, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP). For detection, membrane-bound proteins recognized by the HRP conjugated secondary antibody were visualized by chemiluminescence SuperSignal West Pico ECL Substrate (Thermo Scientific). The films were scanned, and images were analyzed using the image processing software Quantity One (Bio-Rad, UK).

Statistical Analyses

Statistical analyses were conducted using the program GraphPad Prism v5.0 (GraphPad Software). All data were expressed as the mean±SD. The results were considered statistically significant if P-value<0.05. Comparisons among multiple groups were performed using repeated measures analysis of variance (ANOVA) and LSD-test. The Student's t test was carried out in the Western blotting data analysis.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Spatial Learning and Memory Ability in Male and Female Rats

As shown by MWM tests, male and female rats exhibited no difference in locomotor activities during the first training day (P=0.72, n.s.). When tested in the hidden platform water maze task, both sexes displayed a significant decrease in the latency (repeated measures ANOVA, P=0.0009). This indicated the rats were able to locate the platform more effectively with increased training regardless of their sexes. Moreover, the male rats were observed to take less time to find the hidden platform than female rats (repeated measures ANOVA, P=0.01882) (Fig. 1A).

image

Figure 1. Performance of male and female rats in the MWM task. (A) The escape latency of testing performance in male and female rats (P<0.05); (B) Frequencies of platform crossings for male and female rats during the probe trials on the last training day. Results are denoted as average±SD. repeated measures ANOVA, Bonferroni post hoc test (A) and t-test (B).

Download figure to PowerPoint

2DE Analysis of Differentially Expressed Hippocampal Proteins Between Male and Female Rats

One major objective of this study was to investigate the differentially expressed hippocampal proteins in rats of different sexes. The high-resolution 2DE maps were constructed from male and female rats' hippocampi after the MWM training task. For each sex group, a sample was prepared by mixing hippocampi lysates of four individuals. Each sample was subjected to triplicate runs, and the results were highly reproducible. In all, 650±43 and 760±56 spots (Mean±SD; pH 3–10) were observed on the 2DE gels of female and male rats' hippocampi, respectively (Fig. 2). Comparative analyses of each sex's 2DE gels detected 33 protein spots, which were significantly differentially expressed between male and female rats' hippocampal 2DE gels (Fold change>2; P<0.01; Fig. 3). Fourteen protein spots were significantly upregulated in the female rat hippocampal group (Fold change>2; P<0.01) (Fig. 3), and 19 protein spots in male rat hippocampal group (Fold change>2; P<0.01; Fig. 3).

image

Figure 2. The representative 2DE map of the hippocampi of female (F) and male (M) rats. The isolated spots of three most significantly differentially expressed proteins (1 as Spots 116, 2 as Spots 208, and 3 as Spots 702) between two sex groups were identified using MALDI-TOF/TOF MS, as listed in Table 1.

Download figure to PowerPoint

image

Figure 3. Fold change (>2) of significant differentially expressed protein spots (P<0.01) between male and female rats' hippocampi revealed by 2DE gel analysis. 2 A: Fold change of 14 significant differentially expressed proteins (P<0.01) which was upregulated in female rats' hippocampi; 2B: Fold change of 19 significant differentially expressed protein (P<0.01) which was upregulated in male rats' hippocampi.

Download figure to PowerPoint

Identification of Differentially Expressed Proteins

The proteins that showed significant sex-dependent variation in expression were trypsin-digested and analyzed by MALDI-TOF MS. The most significantly upregulated expressed proteins (male: spots 208 and 116; female: spot 702) were further gel-excised and trypsin-digested (Fig. 2). We analyzed the trypsin digest extracts with mass spectrometry, and identified spot 208 as CKB, spot 116 as DRP-2, and spot 702 as GFAP. All identified proteins, including their spectra and molecular weights, are summarized in the Supporting Information Table 1.

Table 1. Three most significantly differentially expressed proteins between male and female rats' hippocampi as identified by peptide mass fingerprint using MALDI-TOF and Mascot Search
Spot No./Spot IDProteinsaNCBInr Acc. No.Calc. MW/Obs. MW (kDa)bCalc. Pl/Obs. PlcMascot scoreFold ratio (male/female)d
  1. a

    DRP-2, CKB and GFAP denote dihydropyrimidinase-related protein 2, creatine kinase B-type, and glial fibrillary acidic protein, respectively.

  2. b

    Calculated molecular weight/observed molecular weight.

  3. c

    Calculated Pl/observed Pl.

  4. d

    Fold ratio (male/female) denotes the relative protein expression volume of male/female.

1/116DRP-2gi¦4025459562/695.95/6.67117103
2/208CKBgi¦20347641/425.39/5.88113323
3/702GFAPgi¦15818673249/595.72/5.71187−143

Validation of the Differentially Expressed Proteins

Protein lysates from four separate male and four separate female rats' hippocampi were resolved by gel electrophoresis, and blotted with the appropriate dilution of antibody (as described in the “Materials and methods” section). The identified proteins included the creatine kinase B-type (CKB), DRP-2, and GFAP (Fig. 4). In line with the 2DE analysis, the Western blotting analysis showed that GFAP was overexpressed in female rats' hippocampi, and CKB and DRP-2 in male rats' hippocampi. The β-actin was equally expressed in the hippocampi of both sexes.

image

Figure 4. Western blotting analyses of partial proteins as revealed by the 2D gel electrophoreses. (A) The detection of DRP-2, GFAP and CKB expression in four male and four female rats' hippocampi; (B) Relative protein band densities of DRP-2, GFAP and CKB. Results are shown as average±SD, n=4, Statistical significance was determined by t-test, where P-values are *<0.05 or **<0.01.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

The biggest challenge to the study of learning and memory lies in that it can never be directly observed and therefore remains a theoretical construct. A task that has been developed to study learning in animals is the Morris water escape task (i.e., the MWM). And in its most basic form, it assesses spatial learning and memory abilities. These abilities are critical for survival, since they enable an individual to find and later remember the location of resources such as food and mates, and avoid predators (Perdue et al., 2009). This task uses a round water pool, in which a platform is submerged beneath the surface. When placed in the maze, the animal's task is to find the hidden platform. The experimenter can chart the “learning” of the animal by the time it takes to find the platform over a number of trials. Since Maccoby and Jacklin (1974) first concluded that males generally outperform females in spatial ability, this finding has been consistently replicated in an abundance of studies spanning multiple developmental phases and spatial tasks (Lewin et al., 2001; Driscoll et al., 2005; Andreano and Cahill, 2009). In this study, MWM task revealed that compared with female rats, male rats could find the hidden platform more efficiently and cross it more frequently. This indicated that males are more excellent at spatial learning and memory than females, in line with previous studies on intersexual differences in such abilities (Lewin et al., 2001; Driscoll et al., 2005; Andreano and Cahill, 2009; Perdue et al., 2009).

In this study, we employed proteomic analyses based on a combined approach of 2DE and MS, to examine differential protein expression in the hippocampi of male and female rats. The result demonstrated quantitative differences in 33 spots between the male and female rats' hippocampi. In all, three differentially expressed proteins were identified and validated, that is, creatine kinase B-type (CKB), DRP-2 and GFAP). We detected higher abundance of both CKB and DRP-2 in the male rats' hippocampi, while the GFAP were overexpressed in the female rats.

GAFP is a cell-specific marker that can distinguishes astrocytes from other glial cells during the development of the central nervous system. It is a marker of mature astrocytes surrounding the synapses and some of their processes interacting with synapses, and is very important for synaptic plasticity (Fields and Stevens-Graham, 2002). Previous studies showed that the expression of GFAP as well as GFAP isotypes may be related to memory retention in rats (Valles et al., 1997; Zhou et al., 2000); and, higher levels of GFAP have been observed in the AD and DS brains (Thompson et al., 2003). Arias et al. (2009) analyzed GFAP immunoreactivity in the hippocampus of intact adult male rats as well as in females during diestrus and proestrus phases of the estrous cycle. They found that in CA1, CA3, and dentate gyrus, GFAP immunoreactivity was higher in proestrus females as compared with males and diestrus females. In CA1, a similar GFAP immunoreactivity was found in males and in diestrus females, while in dentate gyrus, males presented the lowest GFAP content. Thus, in this study, the overexpression of GFAP in female rats' hippocampi was in accordance with the result of the MWM task.

DRP-2 is highly expressed in the brain, is abundant in nervous system especially during prenatal development, and is crucial for axonal outgrowth and determination of the fate of the axon and dendrites, possibly by promoting neurite elongation and branching via microtubule assembly (Byk et al., 1995; Goshima et al., 1995; Minturn et al., 1995; Naoyuki et al., 2001). DRP-2 is enriched in the postsynaptic density, and is also involved in regulating the dynamics of microtubules (Gu and Ihara, 2000; Kawano et al., 2005; Ujike et al., 2006). DRP-2 has been found dysregulated at the protein level in DS and AD brains (Sultana et al., 2007).

This study also detected stronger expression of creatine kinase (CKB) in male rats' than in female rats' hippocampi. CKB is an essential enzyme in energy-hungry tissues such as the brain, in which it catalyses the phosphorylation of creatine to phosphocreatine. CKB is expressed in a variety of tissues, and is most abundant in adult brains where most CKB has been shown to be cytosolic (Urdal et al., 1983; Mitchell and Benfield, 1990; Manos et al., 1991; Friedman and Roberts, 1994; Kaldis et al., 1996; Shen et al., 2002). It implies that the increased expression of CKB may be attributed to the amount of matured neurons in male rats' hippocampi.

To our knowledge, this study represents the first comparative proteomic analysis of hippocampi between male and female rats. The MWM analysis indicated that male rats are more excellent in spatial working and memory abilities. Our proteomic study detected 33 proteins which were differentially expressed between male and female rats' hippocampi. The proteomic identification showed the overexpression of GFAP in female rats' hippocampi, and the overexpression of CKB and DRP-2 in male rats' hippocampi, which confirmed the Western blotting validation. The intersexual differences in hippocampal proteomic profiling may shed light on those in spatial learning and memory abilities. However, due to the limited number of the identified hippocampal proteins, the present study should be considered preliminary. Further efforts would be made to enlarge the number of differently expressed 2D spots and identify more hippocampal proteins, which would enrich our knowledge about the relationship between the intersexual differences in spatial learning and memory abilities and those in the hippocampal protein expression.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED