extfieldsofstudy: Biology Medicine

text:
Histone Hyperacetylation as a Response to Global Brain Ischemia Associated with Hyperhomocysteinemia in Rats

Epigenetic regulations play an important role in both normal and pathological conditions of an organism, and are influenced by various exogenous and endogenous factors. Hyperhomocysteinemia (hHcy), as a risk factor for several pathological conditions affecting the central nervous system, is supposed to alter the epigenetic signature of the given tissue, which therefore worsens the subsequent damage. To investigate the effect of hHcy in combination with ischemia-reperfusion injury (IRI) and histone acetylation, we used the hHcy animal model of global forebrain ischemia in rats. Cresyl violet staining showed massive neural disintegration in the M1 (primary motor cortex) region as well as in the CA1 (cornu ammonis 1) area of the hippocampus induced by IRI. Neural loss was significantly higher in the group with induced hHcy. Moreover, immunohistochemistry and Western blot analysis of the brain cortex showed prominent changes in the acetylation of histones H3 and H4, at lysine 9 and 12, respectively, as a result of IRI and induced hHcy. It seems that the differences in histone acetylation patterns in the cortical region have a preferred role in pathological processes induced by IRI associated with hHcy and could be considered in therapeutic strategies.

Introduction
The epigenetic mechanism of gene regulation in brain tissue has been intensively studied under various physiological and pathological conditions [1,2]. Epigenetic marks represent group of processes and structures that alter gene expression by regulating DNA accessibility. These traits are heritable and do not directly affect the DNA sequence. Epigenetic modifications including DNA methylation, posttranslational modification of histones or noncoding RNA interactions play a pivotal role in a plethora of physiological functions and, remarkably, in many disorders [3,4].
Aberrant regulation of homocysteine (Hcy) metabolism is a sulfur-containing amino acid derived from methionine that leads to the hyperhomocysteinemia (hHcy). In addition to the pleiotropic biochemical properties of Hcy, it can also act through epigenetic pathways by alteration in DNA Int. J. Mol. Sci. 2018, 19, 3147 2 of 15 methylation as a result of imbalance in the levels of biochemical components of Hcy cycle [5][6][7]. In the process of Hcy to methionine conversion S-adenosylmethionine (SAM) arises, a major methyl donor for all methylation reactions within the cell. SAM is further methylated to S-adenosylhomocysteine (SAH). The methylation potential of a cell can be defined by the SAM:SAH ratio [8,9], which is remarkably decreased in hHcy conditions induced both by the deficiency of vitamin B 6 and B 12 or due to genetic polymorphisms of metabolic genes [10]. Several studies support the evidence that hHcy can lead to global DNA hypomethylation [11][12][13][14]. Remarkably, hypomethylation is associated with reduced recruitment of histone deacetylases [15]. This in turn causes accumulation of acetylated H3 and H4 histones at lysine residues [16]. DNA hypomethylation and histone acetylation are associated with a transcriptionally permissive state of chromatin, although the open chromatin state is more available for protein molecules that suppress gene expression. Conversely, Hcy-induced DNA hypomethylation of gene promoter regions results in selective gene upregulation that contributes to augmented tissue injury [17,18].
HHcy is also identified as one of the risk factors and a target of preventive strategies for atherosclerosis, vascular disorders including cerebral stroke, as well as for neurodegenerative diseases and congenital defects [19][20][21]. Besides, these pathological conditions are associated with significant changes in the epigenetic state of the injured organs and the corresponding tissue. For instance, acute cerebral ischemic stroke is related to the increased DNA methylation accompanied with decreased acetylation of histones in neural tissue [22][23][24][25]. The reports on effects of hypoxia/ischemia upon levels of acetylated histones in hHcy conditions in various brain regions are still insufficient. An increasing body of evidence points to the imbalance in histone de/acetylation in different pathologies affecting the central nervous system [26]. Moreover, various gene regions are targeted by different patterns of acetylation resulting in multiple functional modifications. Acetylation of histone H3 at lysine 9 (H3K9ac) is presumably located in the regions surrounding the gene transcriptional start sites, whereas histone H4 lysine 12 acetylation (H4K12ac) is elevated in the promoter and transcribed regions of active genes [27]. This may suggest the different functions of each acetylation pattern. For instance, upregulation of H3K9ac leads to decreased expression of proinflammatory genes [28,29].
Cortical degeneration profoundly affects proper brain function and behavior. Several brain diseases selectively impact specific cortical regions, leaving others relatively intact. For example, Alzheimer's disease (AD) as a severe neurodegenerative disorder, leads to functional deficit and cognitive decline caused by progressive cell loss in certain cortical systems. Among AD, brain cortex is affected in different ways also by other neurological conditions such as schizophrenia or dementia [30]. Our recent studies have shown that global IRI with combination of hHcy leads to more extensive neurodegeneration predominantly in the hippocampus, but to the morphological damage of cortical neurons as well [31,32], in spite of the various vulnerability of neurons to degeneration and apoptosis [33].
Until now, most studies have been focused to the epigenetic effect of Hcy, especially in the relationship with DNA or histone methylation, and not in the association with histone acetylation. Moreover, just a small number of these studies investigate the combination of cerebral IRI and hHcy condition in connection with histone acetylation. In the present study, we attempt to ascertain for the first time the effect of IRI in combination with induced hHcy to examine the extent of neuronal damage and the changes in acetylation level of histones H3 and H4 at specific lysine residues in vulnerable brain areas, such as rat cerebral cortex.

Cresyl Violet Staining
In order to assess the impact of hHcy in association with ischemia and ischemia-reperfusion injury (IRI) to the extent of neuronal damage, we used cresyl violet staining, which is commonly used to identify the neuronal structure in brain and specifically stains Nissl bodies. In the naive control group (C), neural cells in the CA1 and M1 region appeared round with pale stained nuclei and purple Nissl bodies ( Figure 1A,E). Cresyl violet stained rat brain sections and statistical evaluation of changes in number of vital neurons in the CA1 (cornu ammonis 1) region of hippocampus and M1 (primary motor cortex) region of rat brain cortex. Bright-field micrographs of CA1 region of hippocampus representing naive control (A), homocysteine-treated control (Hcy-C) (B), naive ischemia followed by 72 h of reperfusion (IR-72h) (C), Hcy-IR-72h (D) and micrographs of M1 region of rat brain cortex representing naive control (E), Hcy-C (F), naive IR-72h (G) and Hcy-IR-72h (H). Arrows indicate morphologically changed neurons, while arrow heads show vital neurons. Bar = 50 µm, n = 5/group. Schematic coronal rat brain section (according to Paxinos and Watson [34]) showing CA1 area of rat hippocampus (red rectangle) and M1 region (blue rectangle) of cerebral cortex. Number of vital neuronal cells in the CA1 Figure 1. Cresyl violet stained rat brain sections and statistical evaluation of changes in number of vital neurons in the CA1 (cornu ammonis 1) region of hippocampus and M1 (primary motor cortex) region of rat brain cortex. Bright-field micrographs of CA1 region of hippocampus representing naive control (A), homocysteine-treated control (Hcy-C) (B), naive ischemia followed by 72 h of reperfusion (IR-72h) (C), Hcy-IR-72h (D) and micrographs of M1 region of rat brain cortex representing naive control (E), Hcy-C (F), naive IR-72h (G) and Hcy-IR-72h (H). Arrows indicate morphologically changed neurons, while arrow heads show vital neurons. Bar = 50 µm, n = 5/group. Schematic coronal rat brain section (according to Paxinos and Watson [34]) showing CA1 area of rat hippocampus (red rectangle) and M1 region (blue rectangle) of cerebral cortex. Number of vital neuronal cells in the CA1 area of hippocampus (I) and M1 region of rat brain cortex (J) in naive control, Hcy-C, naive IR-72h and Hcy-IR-72h. Results are presented as mean ± SEM, n = 5/group. *** p < 0.001 versus the control value; ++ p < 0.01 and +++ p < 0.001 versus the control with Hcy; & p < 0.05 and &&& p < 0.001 versus the corresponding reperfusion period.
In the Hcy-C group we observed a diminished number of neural cells when compared to the naive control (C), although without notable statistical significance in both CA1 hippocampal and M1 cortical regions ( Figure 1B,F). Conversely, neurons in these brain regions showed obvious morphological changes with signs of swelling and intracellular organelles damage in naive IR-72h group ( Figure 1C,G). These changes were more remarkable in the CA1 hippocampal area where the number of intact neurons decreased significantly to 56% (177.5 ± 9.2; p < 0.001) when compared to the naive control (C) and to 65% in comparison with Hcy-C group (272 ± 17.1; p < 0.01). Neurons in the CA1 region of Hcy-IR-72h group demonstrated more eminent damage then the naive IR-72h group ( Figure 1D). More specifically, we observed massive loss of neurons and total disintegration accompanied by cytoplasm shrinkage and vacuolization. The number of surviving neurons in the Hcy-IR-72h group declined to 38% (120.75 ± 10.9; p < 0.001) when compared to the naive control (C) and to 44% (p < 0.001) in comparison to Hcy-C group in CA1 hippocampal area. Moreover, the number of intact neurons was significantly decreased to 68% (p < 0.05) when compared to the naive IR-72h group ( Figure 1I).
In the Hcy-IR-72h group, neural cells localized in the M1 cortical region showed similar signs of damage ( Figure 1H). Compared to the naive control (C), the number of surviving neurons in this brain area declined to 35% (126.25 ± 20.5; p < 0.001) and to 43% (p < 0.001) in comparison with Hcy-C group. Furthermore, there was a significant decrease of 38% (p < 0.001) between the naive IR-72h and Hcy-IR-72h groups in the M1 cortical region ( Figure 1J).

Immunohistochemical Detection of Acetylated Histone H3 (Lys9) and H4 (Lys12)
After IRI, the histological damage and possible histone posttranslational modifications are first detected and established in the hippocampus where they are transient, and later can also be detected in the cortex [1]. Therefore, as next we investigated the alterations in histone acetylation levels in M1 cortical region. Brain sections were subjected to immunohistochemical analysis to detect acetylated histone H3 at lysine 9 (H3K9ac) and histone H4 at lysine 12 (H4K12ac). The figures show representative pictures from the naive control (C) and Hcy-C group, ischemic groups (ISCH; Hcy-ISCH) and IRI groups with 72 h reperfusion (IR-72h; Hcy-IR-72h) (Figures 2 and 3). area of hippocampus (I) and M1 region of rat brain cortex (J) in naive control, Hcy-C, naive IR-72h and Hcy-IR-72h. Results are presented as mean ± SEM, n = 5/group. *** p < 0.001 versus the control value; ++ p < 0.01 and +++ p < 0.001 versus the control with Hcy; & p < 0.05 and &&& p < 0.001 versus the corresponding reperfusion period.
In the Hcy-C group we observed a diminished number of neural cells when compared to the naive control (C), although without notable statistical significance in both CA1 hippocampal and M1 cortical regions ( Figure 1B,F). Conversely, neurons in these brain regions showed obvious morphological changes with signs of swelling and intracellular organelles damage in naive IR-72h group ( Figure 1C,G). These changes were more remarkable in the CA1 hippocampal area where the number of intact neurons decreased significantly to 56% (177.5 ± 9.2; p < 0.001) when compared to the naive control (C) and to 65% in comparison with Hcy-C group (272 ± 17.1; p < 0.01). Neurons in the CA1 region of Hcy-IR-72h group demonstrated more eminent damage then the naive IR-72h group ( Figure 1D). More specifically, we observed massive loss of neurons and total disintegration accompanied by cytoplasm shrinkage and vacuolization. The number of surviving neurons in the Hcy-IR-72h group declined to 38% (120.75 ± 10.9; p < 0.001) when compared to the naive control (C) and to 44% (p < 0.001) in comparison to Hcy-C group in CA1 hippocampal area. Moreover, the number of intact neurons was significantly decreased to 68% (p < 0.05) when compared to the naive IR-72h group ( Figure 1I).
In the Hcy-IR-72h group, neural cells localized in the M1 cortical region showed similar signs of damage ( Figure 1H). Compared to the naive control (C), the number of surviving neurons in this brain area declined to 35% (126.25 ± 20.5; p < 0.001) and to 43% (p < 0.001) in comparison with Hcy-C group. Furthermore, there was a significant decrease of 38% (p < 0.001) between the naive IR-72h and Hcy-IR-72h groups in the M1 cortical region ( Figure 1J).
It can be seen from the micrographs that induction of hHcy significantly affected the histone acetylation status in the M1 cortical region, which is obvious by the increased number H3K9ac+ cells ( Figure 2G). Moreover, H3K9ac+ cells between the naive ISCH and Hcy-ISCH group were elevated to 841% (7.25 ± 1.7; p < 0.001) and when comparing naive and Hcy-treated IR-72h groups decreased to 66% (p < 0.001). Additionally, the elevated H3K9ac immunopositivity in both IR-72h groups was statistically significant in the naive ISCH (p < 0.001) and Hcy-ISCH group (p < 0.001).
In contrast, the differences in the levels of H4K12ac in cortex between the naive and Hcy-treated groups were less prominent, but still significant. The highest H4K12ac immunopositivity was observed in the naive control (C) and Hcy-C group ( Figure 3A-F). Ischemic insult resulted in a Results are presented as mean ± SEM, n = 5/group. * p < 0.05 and *** p < 0.001 versus the control value; + p < 0.05, ++ p < 0.01 and +++ p < 0.001 versus the control with Hcy; •• p < 0.01 versus the naive ISCH group; & p < 0.05 and && p < 0.01 versus the corresponding reperfusion period.
Signals corresponding to H3K9ac and H4K12ac were predominantly located within the nuclei. There were obvious changes in histone acetylation in all experimental conditions, with remarkable differences between the naive and Hcy-groups. As seen from the fluorescent micrographs, the lowest H3K9ac immunopositivity (+) was detected in the naive ISCH group and in the naive control (Figure 2A-F). Compared to the naive control (C), the number of H3K9ac immunopositive cells in naive ISCH group decreased, although not significantly, but highly increased to 716% (145 ± 6.5; p < 0.001) in the naive IR-72h group. The number of H3K9ac+ cells in the Hcy-C group was elevated to 607% (123 ± 2.4; p < 0.001), in the Hcy-ISCH group to 301% (61 ± 4.7; p < 0.001) and the increase was up to 472% (95.5 ± 4.7; p < 0.001) in the Hcy-IR-72h group against the naive control (C). H3K9+ cells in Hcy-ISCH group declined by 50% (p < 0.001) and by 78% (p < 0.01) in Hcy-IR-72h group when compared to the Hcy-C group.
It can be seen from the micrographs that induction of hHcy significantly affected the histone acetylation status in the M1 cortical region, which is obvious by the increased number H3K9ac+ cells ( Figure 2G). Moreover, H3K9ac+ cells between the naive ISCH and Hcy-ISCH group were elevated to 841% (7.25 ± 1.7; p < 0.001) and when comparing naive and Hcy-treated IR-72h groups decreased to 66% (p < 0.001). Additionally, the elevated H3K9ac immunopositivity in both IR-72h groups was statistically significant in the naive ISCH (p < 0.001) and Hcy-ISCH group (p < 0.001).
In contrast, the differences in the levels of H4K12ac in cortex between the naive and Hcy-treated groups were less prominent, but still significant. The highest H4K12ac immunopositivity was observed in the naive control (C) and Hcy-C group ( Figure 3A-F). Ischemic insult resulted in a significant decline in the number of H4K12ac+ cells to 52% (65 ± 6.4; p < 0.001) in the naive ISCH group when compared to the naive control (C). The elevation in immunolabelled H4K12ac cells in the Hcy-C group to 118% (147.5 ± 11; p < 0.05) was also statistically significant when compared to the naive control (C). Ischemia and IRI induced significant decline in the number of H4K12+ cell in the naive ISCH group (44%; p < 0.001) and in the naive IR-72h to 64% (94 ± 5.2; p < 0.01) compared to the Hcy-C group. The decrease by 75% (110 ± 7.2; p < 0.05) between the Hcy-C and Hcy-ISCH group was also statistically significant. Furthermore, the increase by 169% (p < 0.01) between the Hcy-ISCH and naive ISCH group was significant, as well as the 128% elevation (120 ± 3.9; p < 0.05) among the Hcy-IR-72h and naive IR-72h groups. In addition, the difference in H4K12ac+ cells in naive IR-72h group compared to naive IR-72h was significantly elevated, too ( Figure 3G).

Detection of H3K9ac and H4K12ac Levels in Cortical Homogenates
To support the above-mentioned immunohistochemical results, the changes in histone acetylation in the rat brain cortex in all experimental groups were also examined by Western blot analysis. We documented similar differences in histone acetylation in cortical tissue homogenates. Hyperacetylation of H3K9 was the most significant after the IRI in the naive IR-72h group (3422%; p < 0.001) when compared to the naive control (C). Surprisingly, the combination of IRI and induced hHcy in the Hcy-IR-72h group resulted in the increase of H3K9ac up to only 1316% (p < 0.05) in comparison to naive control (C), which declined markedly by 2.6-times (p < 0.001) in comparison to naive IR-72h group ( Figure 4A). significant decline in the number of H4K12ac+ cells to 52% (65 ± 6.4; p < 0.001) in the naive ISCH group when compared to the naive control (C). The elevation in immunolabelled H4K12ac cells in the Hcy-C group to 118% (147.5 ± 11; p < 0.05) was also statistically significant when compared to the naive control (C). Ischemia and IRI induced significant decline in the number of H4K12+ cell in the naive ISCH group (44%; p < 0.001) and in the naive IR-72h to 64% (94 ± 5.2; p < 0.01) compared to the Hcy-C group. The decrease by 75% (110 ± 7.2; p < 0.05) between the Hcy-C and Hcy-ISCH group was also statistically significant. Furthermore, the increase by 169% (p < 0.01) between the Hcy-ISCH and naive ISCH group was significant, as well as the 128% elevation (120 ± 3.9; p < 0.05) among the Hcy-IR-72h and naive IR-72h groups. In addition, the difference in H4K12ac+ cells in naive IR-72h group compared to naive IR-72h was significantly elevated, too ( Figure 3G).

Detection of H3K9ac and H4K12ac Levels in Cortical Homogenates
To support the above-mentioned immunohistochemical results, the changes in histone acetylation in the rat brain cortex in all experimental groups were also examined by Western blot analysis. We documented similar differences in histone acetylation in cortical tissue homogenates. Hyperacetylation of H3K9 was the most significant after the IRI in the naive IR-72h group (3422%; p < 0.001) when compared to the naive control (C). Surprisingly, the combination of IRI and induced hHcy in the Hcy-IR-72h group resulted in the increase of H3K9ac up to only 1316% (p < 0.05) in comparison to naive control (C), which declined markedly by 2.6-times (p < 0.001) in comparison to naive IR-72h group ( Figure 4A). . Western blot analysis of H3K9ac (A) and H4K12ac (B) levels in rat brain cortex homogenates. Levels of histones acetylation in the M1 area in naive control, Hcy-C, naive ISCH, Hcy-ISCH, naive IR-72h and Hcy-IR-72h. Results are presented as mean ± SEM, n = 5/group, normalized to the control levels. * p < 0.05 and *** p < 0.001 versus the control value; ++ p < 0.01 and +++ p < 0.001 versus the control with Hcy; • p < 0.05 and ••• p < 0.001 versus the naive ISCH group; ◊◊◊ p < 0.001 versus the ISCH with Hcy; && p < 0.01 and &&& p < 0.001 versus the corresponding reperfusion period.
Regarding the levels of H4K12ac in cortex, the combination of Hcy treatment and IRI in the Hcy-IR-72h group resulted to a significant elevation by 2.4-times (p < 0.001) when compared to the naive control (C). In the naive ISCH group, level of H4K12ac was 6.26-times lower (p < 0.05) than the naive control (C) and 7-times lower (p < 0.01) than in the Hcy-C group. Remarkably, the elevation of H4K12ac in Hcy-ISCH group almost 8-times (p < 0.01) was significant when compared to the naive ISCH group, as the detected level of H4K12ac was nearly 3-times higher between the naive IR-72h and Hcy-IR-72h group (p < 0.001). Nevertheless, we observed markedly increased H3K9ac level . Western blot analysis of H3K9ac (A) and H4K12ac (B) levels in rat brain cortex homogenates. Levels of histones acetylation in the M1 area in naive control, Hcy-C, naive ISCH, Hcy-ISCH, naive IR-72h and Hcy-IR-72h. Results are presented as mean ± SEM, n = 5/group, normalized to the control levels. * p < 0.05 and *** p < 0.001 versus the control value; ++ p < 0.01 and +++ p < 0.001 versus the control with Hcy; • p < 0.05 and ••• p < 0.001 versus the naive ISCH group; ♦♦♦ p < 0.001 versus the ISCH with Hcy; && p < 0.01 and &&& p < 0.001 versus the corresponding reperfusion period.
Regarding the levels of H4K12ac in cortex, the combination of Hcy treatment and IRI in the Hcy-IR-72h group resulted to a significant elevation by 2.4-times (p < 0.001) when compared to the naive control (C). In the naive ISCH group, level of H4K12ac was 6.26-times lower (p < 0.05) than the naive control (C) and 7-times lower (p < 0.01) than in the Hcy-C group. Remarkably, the elevation of H4K12ac in Hcy-ISCH group almost 8-times (p < 0.01) was significant when compared to the naive ISCH group, as the detected level of H4K12ac was nearly 3-times higher between the naive IR-72h and Hcy-IR-72h group (p < 0.001). Nevertheless, we observed markedly increased H3K9ac level between the naive control (C) and Hcy-C group, as well as a smaller increase in H4K12ac, none of these changes observed by Western blot were statistically significant ( Figure 4B).
Based on our morphological and Western blot observations, we could assume that the combination of IRI with hHcy triggers different responses of H3K9 and H4K12 histone acetylation patterns in the M1 cortical region. This conclusively suggest that hHcy conditions exaggerate processes linked with neuronal damage and epigenetic changes are likely to participate in the mechanism of neurodegeneration induced by IRI and hHcy.

Discussion
Despite numerous studies devoted to the explanation of stroke etiology, we still have only limited knowledge describing the complex mechanisms of neuronal damage. It is well known that ischemia induces significant changes in the expression of regulatory genes within the biological response to injury, but it also triggers alterations in the epigenetic patterns of the injured organ [35][36][37]. In spite of the known epigenetic influence in the development of various pathological states, only limited studies can be found that describe histone acetylation after IRI under hHcy conditions. Thus, the aim of the present study was to determine how IRI in combination with hHcy affects the extent of neurodegeneration and alters histone acetylation patterns in brain cortex.
Ischemic insult initiates disruption of the blood-brain barrier (BBB) that allows for easier penetration of toxicants such as Hcy [38]. Hcy in turn can induce vulnerable tissue damage, predominantly in the cerebral cortex and hippocampus [31,33]. Moreover, it can cause cellular damage by altering the subtle epigenetic state of all neural cells such as neurons, astrocytes and microglia in vulnerable areas [39].
In this paper, we first focused on the analysis of morphological changes after ischemic insult. Similar to our previous studies [31,32], the most prominent disintegration of neural cells was detected in the naive IR-72h group in the CA1 area of hippocampus and cortical region, and hHcy conditions in the Hcy-IR-72h group exaggerate the extent of this neuronal damage.
Several experimental approaches proved that disarrangement of histone acetylation can contribute to the processes linked with neuronal death. Selective histone acetylation is linked with protection of neurons against oxidative injury by enhanced gene regulation of antioxidant enzymes participating in the neuroprotection [40]. In general, tissue ischemia is associated with the rise in a global amount of DNA-and histone methylation, and this increase correlates with the extension of brain injury [22,23]. However, this effect is tissue and gene specific [41]. Conversely, global hypoacetylation of histones H3 and H4 correlates with ischemic/hypoxic conditions [42,43]. However, some studies observed increased levels of histone acetylation after a preconditioning-tolerance building stimuli [43]. Numerous results of experiments demonstrated a massive decrease in the level of histone acetylation in the ischemic brain persisting up to for 2 weeks after the stroke [44,45]. Interestingly, the histone deacetylase activity does not change during the first 6 h after ischemia and it is upregulated within 7 days after the stroke, which corresponds with the period of the partial recovery of mostly penumbral region after injury [24,26]. Higher level of plasma Hcy interferes with the neuronal metabolism in the brain parenchyma. There is no straightforward evidence for the direct Hcy inclusion in the histone acetylation, it changes the methylation levels and subsequently alters the activity of methyl-binding proteins. This leads to the formation of complexes with histone deacetylases and consequently to the posttranslational histone modifications [14][15][16].
Our paper describes, for the first time, how ischemic insult in combination with hHcy affects the H3 and H4 histone acetylation pattern at specific lysine residues in vulnerable brain area. Generally, as seen from our results, the cortical tissue damage in IR-72h group combined with induced hHcy is linked with the changes in acetylation level of histones and responds to the degree of neuronal damage. Moreover, hHcy condition alone triggers changes at the acetylation process, likely within the context of hypomethylation due to the elevated level of Hcy.
Results of the study of Jin et al. [39] documented global H3K9 hyperacetylation in combination with global hypomethylation in cultured rat astrocytes after Hcy exposure. This leans towards the altered regulation of gene expression following Hcy treatment. Apparently, an epigenetic mechanism might regulate Hcy-related metabolic changes including mitochondrial function. Furthermore, another link between the increased Hcy level and histone acetylation was observed by Xu et al. [46] by the elevated level of H3 acetylation in the presence of its metabolite Hcy-thiolactone.
According to the recent study of Patnala et al. [28], global upregulation of H3K9ac is a consistent feature of activated microglia in the ischemic region. Furthermore, these gene-specific H3K9ac modulations may alter the expression of pro-and anti-inflammatory genes in microglia of ischemized tissue. Similar changes were documented in long-term in vitro Hcy-treated microglia by the reduced pro-and increased anti-inflammatory markers after 72 h of Hcy exposure [47]. Besides, more proinflammatory phenotype was manifested in a hHcy model in mice induced by chronic hHcy diet [48,49].
A global transcriptional repression induced by ischemia is reflected by the alterations of histone H3 and H4 in the injured area [50,51]. This is reasonable, since acetyl-CoA, the fundamental cofactor for histone acetyltransferases (HAT), is the only substrate for acetylation reactions. Its supply is in hypoxic/ischemic conditions depleted due to the inhibition of pyruvate dehydrogenase complex [52][53][54] which might result in the reduced activity of HAT. Moreover, several papers point to the connection between increased oxidative stress, resulting in neurotoxicity and changes in histone modifications/acetylation [55][56][57][58][59]. Oxidative stress causes DNA damage, heterochromatin loss and subsequent aberrant gene expression, inappropriate cell cycle activation and neuronal apoptosis. In the light of these findings there is a possibility that these processes relate to other neurodegenerative diseases with a link to oxidative stress [57]. Hcy acts as an excitatory amino acid that increases unregulated reactive oxygen species (ROS) production and elevates susceptibility of neurons to oxidative stress. The brain possesses fewer antioxidant enzymes than other body tissues and thus these conditions make the brain more vulnerable to oxidative stress and initiate neuronal cell death. Microvasculature and the BBB are also affected by hHcy, which are strictly controlled in the cerebral cortex [60].
Many papers described disarrangement in histone acetylation levels also in the neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Interestingly, the hHcy and ischemic/reperfusion damage are recognized as independent risks in these pathologies. Redistribution of acetylation patterns has also been characterized as age-dependent. Nativio et al. [61] observed increased histone acetylation in aged human controls, but decreased acetylation in comparison with AD patients. Hyperacetylation of H3K9 was also observed in the post-mortem brain of AD patients by Lithner et al. [62]. It has been also pointed out that the global increase in histone H3 acetylation is fundamental in amyloid-related pathogenesis and the formation of AD [62,63]. In addition, Narayan et al. [64] found markedly increased acetylation of H3 and H4 histones along with increased total histone levels in the regions of high pathology in post-mortem AD brains and suggest that any acetylation changes could be due to the changes in the total histone protein levels. Remarkably, the cardiac arrest ischemia is likely to induce genes and proteins connected with AD, contributing to neuronal cell death and neurodegeneration of cerebral tissue [65].
Finally, we could only hypothesize about the effect of how altered acetylation of H3K9 and H4K12 participate in the tissue response in the hHcy IRI animal model. The elevated level of H3K9ac in reperfusion period may be due to the activated expression of genes contributing in the response mechanisms initiated by hHcy and ischemic insult. The elevated acetylation in the hHcy controls may be implicated with known inflammatory responses induced by Hcy [5,7,9]. Changes in acetylation patterns of H3K9 and H4K12 might also be a result of depleted acetyl-CoA required for the acetylation process and the Hcy-induced oxidative stress, followed by the ischemic insult.
In summary, our results contribute to the understanding of how ischemic insult, which is combined with recognized risk factor-hHcy, induces changes in the epigenetic status, especially histone acetylation in the cells of vulnerable brain areas. These changes might modify transcriptionally active or inactive states of chromatin and gene expression as a part of tissue response to the insult. In fact, certain combinations of acetyl and also methyl post-translational histone tail lysine modifications may have an antagonistic or cooperative biological effects [1,2,50,51]. Until now, only sparse information can be found which describe alterations of the whole epigenetic machinery in a complex event such as cerebral ischemic injury combined with hHcy.

Animals and Experimental Design
Animal studies were approved by the ethics committee of Jessenius Faculty of Medicine, Comenius University (19 August 2016, approval code: EK 1647/2015) and by the State Veterinary and Food Department of the Slovak Republic (no. 2857/16-221) in the grant titled "Epigenetic and molecular mechanisms of neuroprotection and ischemic tolerance". The animals were handled according to the European Union legislation "on the protection of animals used for scientific purposes" (Directive 2010/63/EU). In our study, adult male Wistar rats (Velaz, Prague, Czech Republic) were used, weighing 300-400 g (4-6 months) at the beginning of the experiment with total n = 72. Animals were maintained in air-conditioned rooms with sustained standard conditions (22 ± 2 • C and 12 h day/night cycle). Availability of food and water was ad libitum. Global forebrain ischemia was performed by using standard 4-vessel occlusion model developed by Pulsinelli and Brierley [66]. Briefly, the rats were placed in an anesthetic box and anesthetized by inhalation of sevoflurane in a mixture of 33% O 2 /66% N 2 O (4.5% sevoflurane for induction of anesthesia and then 3-3.5% sevoflurane for anesthesia maintenance. Rectal temperature was preserved at 37-38 • C during the surgery with a heating pad. Firstly, both vertebral arteries were irreversibly occluded (day 1) by thermocoagulation through the alar foramina. On the day 2, both common carotids were occluded in duration of 15 min. The anesthetic conditions were maintained as described above. Approximately 2 min before carotid occlusion, the anesthesia was removed and 15 min of ischemia with 72 h of reperfusion followed.
Mydriasis, loss of the righting reflex and paw extension were criteria for forebrain ischemia. The rats which showed no seizures during and after ischemia and those that became unresponsive, lost the righting reflex during carotid occlusion were implied for this experiment. Due to different methods of preparation of biological material for biochemical and histological analysis, 10 animals/experimental group were used (5 animals/method of preparation). After ischemia, animals were sacrificed by decapitation or perfusion (depending on the type of proceeding method) in a mild sevoflurane anesthesia. Brains were dissected and processed immediately. Naive control group of animals proceeded by the same surgical procedure besides carotid occlusion.

Induction of hHcy
Hcy (Sigma-Aldrich, Bratislava, Slovak Republic) was dissolved and buffered as described in our previous paper by Kovalska et al. [33] in concentration of 1.2 µmol/g of body weight). The Hcy injections were administered subcutaneously once a day for 21 days according to Matté et al. [67]. Immediately, six experimental groups were established at day 22. It is well-known that Hcy crosses the BBB and presents a maximal level in the brain 15-60 min after Hcy injection [38,67]. Selected doses of Hcy corresponded to pharmacokinetic parameters, as described in the work of Martins et al. [68]. The level of plasma Hcy in rats reached similar concentrations as referred in previous papers from our laboratory [31][32][33]69].

Cresyl Violet Staining
The above-mentioned groups (groups 1, 2, 5 and 6) of animals (n = 5/group) were anesthetized by inhalation of sevoflurane in a mixture of 33% O 2 /66% N 2 O (4.5% sevoflurane for induction of anesthesia and then 3-3.5% sevoflurane for anesthesia maintenance). Following the anesthesia, the rats underwent a transcardial perfusion with 0.1 mol/L phosphate-buffered saline and 4% paraformaldehyde in 0.1 mol/L PBS [31,70]. After decapitation, the brains were removed from the skull, immersed overnight in the same fixative at 4 • C and placed in 30% sucrose for 24 h. Samples were covered with embedding medium (Killik, Bio Optika, Milano, Italy) and immediately frozen by rapid cooling boost in a cryobar (Shannon Cryotome E, Thermo Scientific, Waltham, MA, USA). Serial coronal 30 µm frozen sections were cut, mounted into Superfrost Plus glass (Thermo Fisher Scientific, Dreieich, Germany) and air-dried. Sections were dehydrated through descending grades of ethanol (95%, 70%, 50%, respectively) and brought to distilled water. The slides were then immersed in cresyl violet staining solution (Lachema, Brno, Czech Republic) for 20 min and washed again in distilled water. After dehydration through the ascending grades of ethanol, the sections were immersed two times for 5 min in xylene solution and coverslipped with entellan. The slides were subsequently examined under the Olympus BX41 light microscope (Olympus, Tokyo, Japan). The counting window for detection of neuronal density presented 1 mm 2 . The number of surviving pyramidal cells was calculated in M1 area of cerebral cortex. Neurons were observed in a double-blind manner and counted on three accidental microscopic fields by two observers.

Detection of Acetylated Histones by Fluorescent Immunohistochemistry
Rabbit polyclonal Anti-acetyl-Histone H3 (Lys9) (Merck Millipore, Darmstadt, Germany, 1:100) and Anti-acetyl-Histone H4 (Lys12) (Merck Millipore, Darmstadt, Germany, 1:100) primary antibodies were used for immunofluorescence. Brain sections from groups 1-6 (see Experimental groups of animals) mounted at Superfrost Plus glass (Thermo Fisher Scientific, Dreieich, Germany) were permeabilized with 0.1% Triton X-100, preblocked with 10% BSA for 1 h. Primary antibodies were applied at 1:100 in the 0.1% Triton X-100 solution with 10% BSA for 24 h at 4 • C, followed by washing with PBS at room temperature. Secondary antibodies were applied at 1:100 for 2 h at room temperature. Detection was performed using Alexa Fluor 488 goat anti-rabbit IgG-conjugated secondary antibodies (1:100, Life Technologies, Carlsbad, CA, USA). The slices were washed again and mounted in Fluoromont-G containing 4 ,6-diamidino-2-phenylindole (Southern Biotechnology Associates, Birmingham, AL, USA). We did not find any immunoreactivity in samples without primary antibodies. To minimize the differences between animals, the fluorescent immunohistochemical analysis was accomplished for each time of reperfusion per animal four times. Positive neuronal cells were counted in M1 cortical area by each animal at least in three different sections. The slides were scanned with a confocal laser scanning microscope (Olympus FluoView FV10i, Tokyo, Japan), objective of 10× with zoom up to 40× magnification equipped with filter for Alexa Fluor 488. The images captured with confocal microscope were analyzed with Olympus FluoView FV10-ASW software (version 02.01, Olympus, Tokyo, Japan), Quick Photo Micro software (version 2.3, Promicra, Prague, Czech Republic) and sequentially with Adobe Photoshop CS3 Extended (Adobe System, San Jose, CA, USA).

Quantitative Image Analysis
A total of 270 selected fields of view (average number: 90 fields/staining method and antibody; 3 sections per animal) taken at 40× magnification were analyzed by ImageJ software (National Institute of Health, Bethesda, MD, USA). The counting parameters for cresyl violet, H3K9ac and H4K12ac immunoreactive cells in 3 distinct fields of M1 cortical area of rat brain were elected as follows. The sampling grid size was 0.06 × 0.06 cm and the counting frame size was 0.03 × 0.03 cm. The total number of labelled cells was expressed per mm 2 .

Data Analysis
The values acquired from image analysis were proceeded with GraphPad Prism software (version 6.01 for Windows, La Jolla, CA, USA). Data are presented as the mean ± SEM. The inter-group statistical significance of mean differences was assessed by ANOVA (one-way analysis of variance) followed by a Student-Newman-Keuls test. The means of control (C), hHcy control (Hcy-C), ischemic group (ISCH) and hHcy ischemic group (Hcy-ISCH), IRI group (IR-72h) and hHcy IRI group (Hcy-IR-72h) were compared. The results from Western blot analysis were normalized to naive control which represents 100%. A value of p < 0.05 was considered to be statistically significant.


s2fieldsofstudy:
Biology
Environmental Science

text:
A new species of Euchaetes Harris from southern Arizona (Erebidae, Arctiinae)

Abstract Euchaetesnancyaesp. n. is described from southeastern Arizona. Although superficially similar to species of Pygarctia Grote, structural and molecular variation shows it to be most closely related to Euchaeteshelena (Cassino). Adults, genitalic structure, eggs, and first instar larvae are described and illustrated. The larval host plant remains unknown. Euchaeteshelena is confirmed as occurring in Mexico.

Introduction
The genus Euchaetes Harris currently encompasses 20 species (Vincent and Laguerre 2014;Lafontaine and Schmidt 2010), most being typical of arid and semi-arid habitats of the American Southwest and Mexico. Two species described from Brazil are not congeneric (Watson andGoodger 1986, Hendrickson 2014). There is no modern revision of the genus, although Hendrickson (2014) provides an as yet unpublished revision wherein a number of new generic combinations are proposed. As a revision will require a review of all species in the genus, in addition to the closely related Pygoctenucha Grote and Pygarctia Grote, we place the new species described herein within the current but unrevised concept of Euchaetes, pending an in-depth review of the Euchaetes group of genera. Preliminary phylogenetic analyses by DaCosta et al. (2006) and Hendrickson (2014) indicate that Euchaetes is polyphyletic.

Materials and methods
Genitalic preparation techniques follow Jaeger (2017). Briefly, abdomens were macerated in 10% KOH solution overnight at room temperature, followed by cursory cleaning and separation of the genitalic capsule, and sequential transfer to 50% EtOH, 70% EtOH and 95% isopropanol. Vesica and corpus bursae inflation was carried out in 50% EtOH, followed by transfer to 70% EtOH for staining. Two stains (both in ethanol solution) were used, first chlorazol black (10 sec) then acidified eosin Y (4 + 4 sec in microwave). Stained tissues were dehydrated in 95% isopropanol before slide mounting in Euparal. Genitalia were imaged using a Leica DFC450 camera, Leica Application Suite 4.8 with a Leica M205C stereo microscope, and processed in Adobe PhotoShop. Interpretation of genitalic morphology and associated terminology differs from that of DaCosta et al. (2006) and Hendrickson (2014) in several respects. We consider the female appendix bursae in the sense of DaCosta to actually represent the bulla seminalis (see e.g., Bendib and Minet 1999), whereas the structure termed the "saccus" is the sacculus. Botanical nomenclature follows the PLANTS database (USDA 2018).
Variation of the 'barcode' section of the COI gene of two Euchaetes nancyae specimens was compared to all other known North American Euchaetes species (Zahiri et al. 2017). DNA extraction, PCR amplification, and sequencing of the COI barcode region was performed at the Canadian Centre for DNA Barcoding (CCDB) and followed standard protocols ; http://www.ccdb.ca/resources.php). DNA sequence analysis metrics were obtained from the Barcode of Life web interface. Resulting data were managed and analyzed using BOLD (Barcode of Life Data Systems; http://v4.boldsystems.org/). Mitogenomic divergence was calculated based on Kimura 2-Parameter (K2P) distances of COI barcodes.
Repository abbreviations are as follows:

Results and discussion
Examination of the type specimens of Euchaetes and Pygarctia described from southwestern USA and Mexico (USNM, AMNH and as illustrated in Hendrickson 2014), in addition to comparison of museum material, shows that the species described here does not have any close relatives in either genus. The lack of a prothoracic foretibial claw, and long second segment of the labial palpus places the new species in Euchaetes rather than Pygarctia (Forbes 1960;DaCosta et al. 2006: fig. 3 Etymology. Euchaetes nancyae is named in honor of the senior author's wife who not only has long supported her husband's study of southwest moths but has also played host to numerous visiting lepidopterists. Nancy won a weekend trip to the Hall Ranch, which led to the initial capture and discovery of this species. Diagnosis. Euchaetes nancyae is superficially similar to other species of Euchaetes and Pygarctia that have predominantly white or grey-white wings, but can generally be distinguished by examination of the head and thoracic colour patterns alone, as illustrated in Figs 1-6. Euchaetes helena and Pygarctia flavidorsalis Barnes & McDunnough have yellow instead of pink scaling on the head. The most common look-alike species that is sympatric with E. nancyae is Pygarctia roseicapitis (Neumögen & Dyar), but E. nancyae differs most obviously from that species by the white (Figure 1   a white abdominal tuft, whereas the tuft is absent in E. nancyae. The distributions of E. nancyae and E. castalla overlap in at least southeastern Arizona (e.g. California Gulch, Pima Co.), and may do so also in northern Mexico. Morphologically, the male uncus and vesica of E. nancyae is highly distinctive, particularly the setation of the lateral lobes and the structure of the dorsomedial lobe, as described below and illustrated in Figs 9, 10. Females differ from those of E. helena in the shape of the cervix bursae and the papillae anales (Figs 11, 12). Structurally, E. nancyae differs from all Pygarctia species by the lack of a foretibial spine and a shorter 2 nd segment of the labial palpus, in addition to the genus traits given below in the Discussion.
DNA barcode sequences of E. nancyae are more than 6% divergent from all other North American Euchaetes, in addition to several undescribed Mexican and Central American species. The two sequenced specimens of E. nancyae differ by 0.46%, forming a unique BIN (Barcode Index Number; Rathnasingham and Hebert 2013). The most similar species based on nearest-neighbour distance analysis is Euchaetes helena, differing by a minimum of 6.61%.
Description. Head. Vestiture of sexes similar; frons and vertex covered with white scales; posterior margin of head with a delicate ring or collar of pale orange-pink scales, extending around posterior and ventral margin of eye; male antenna bipectinate, longest rami 2.5 x longer than antennal segment, scape and proximal third dorsally scaled with white, grading into darker pale grey-brown scaling over distal two-thirds; female antenna biserrate, dorsal vestiture similar to that of male. First segment of labial palpus with relatively long, pale orange-pink scales along ventral edge, second segment covered with white scales, third segment covered with light brown scales; length ratio of segments 1:2:3 = 2.2 : 3.0 : 1.0.
Thorax. Vestiture of sexes similar; patagium, tegula, and thorax covered with greyish-white scales, lateral margin of patagium and tegula with a border of long pale pink hairs extending underneath wings; foreleg coxa pale orange pink with a medial patch of pale grey-brown scales; foretibia without claw at apex; femur and tibia white dorsally, ventrally a mix of white and pale grey-brown scales, tibial spurs and tarsal segments pale grey brown; mid and hind legs with coxa, femur, and tibia covered with a mix of white and pale grey brown scales; metepisternum lacking microtymbals.
Abdomen. Colouration of sexes similar; dorsum pale silvery pinkish orange with a row of 6-7 dorsomedial macules at anterior tergal margin, this dark grey and indistinctly ringed with whitish grey; laterally with a row of prominent black maculae at anterolateral angle; ventrum silvery whitish grey; female without terminal tuft of long scales; intersegmental membrane between sternites 7-8 of male with well-developed coremata, nearly length of abdomen when fully extended.
Male genitalia. Gross morphology typical of Euchaetes, with costa and sacullus deeply divided, costa forming a long prong-like finger, and sacculus forming a mem- branous, elongate lobe; saccus broad and collar like, not tapered to a point medioventrally as in many arctiines; juxta lightly sclerotized and indistinctly differentiated, broadly U-shaped; uncus complex, with a dorsoventrally flattened lateral lobe; each lobe densely setose, forming an evenly sinuate "hair-do" that terminates in an anteriorly-directed, dense, pointed tuft; dorsomedial process of uncus with a pyramidal base, terminating in a laterally flattened crest; apex of uncus laterally flattened and shaped like a broad bird's beak in lateral view; phallus 5 × longer than wide, curved slightly dorsad; vesica roughly kidney shaped with small subbasal diverticulum; distomedial row of approximately 20 sawtooth-like cornuti, decreasing in size towards ductus ejaculatorius.
Female genitalia. Distal margin of papillae anales irregular rounded quadrate; papillae anales moderately setose, densely and finely setose dorsally below apertures of dorsal pheromone gland; pseudopapillae anales membranous; dorsal pheromone gland unbranched, terminating as pair of openings; length of anterior and posterior apophyses approximately equal to maximum width of papillae; cervix bursae moderately sclerotized, internally rugose near junction with ductus, remainder with regularly spaced microtrichia or spicules; ductus bursa flattened dorsoventrally and ribbon-like; corpus bursa essentially spherical, with two star-like signa, one dorsal and one ventral; ventral signum with four arms or rays, dorsal one with three.
Biology and distribution. The larval hostplants of E. nancyae are unknown. Two females oviposited approximately 30 shiny yellow-white eggs measuring 0.8 mm in diameter. Euchaetes nancyae lacks female abdominal tufts, and eggs were not covered with abdominal setae. Presumably, all Euchaetes species that possess female abdominal tufts cover their eggs, such as E. egle (Drury). The first instar larvae were offered several species of Asclepiadaceae including Funastrum cynanchoides (Decne.) Schltr. and one species of Euphorbiaceae, Chamaesyce hyssopifolia (L.), but refused to feed and perished. The related species Euchaetes zella and E. perlevis feed on Funastrum cynanchoides, and Euchaetes fusca larvae feed on the Cnidoscolus angustidens Torr. (Euphorbiaceae). The larval foodplant of the probable sister species Euchaetes helena from Texas is also unknown, but larvae of that species also do not accept Asclepiadaceae (D. Wagner, pers. comm.).
Euchaetes nancyae is known only from the type series, collected in Sonoran desert habitat along the Mexican border in Santa Cruz County, Arizona. It undoubtedly also occurs in adjacent parts of Mexico. Euchaetes helena occurs in Texas (Davis Mountains), and was listed as likely occurring in Mexico by Vincent and Laguerre (2014); we here confirm the occurrence of E. helena from Coahuila, verified by DNA barcode sequence (DNA vouchers CNCLEP 00113425 and CNCLEP00113424).

Conclusion
Based on morphological similarities, Euchaetes nancyae is most closely related to E. helena, E. zella, E. perlevis and E. fusca. Species in this group all share 1) an untufted female abdomen (tufted in other North American species), 2) a well-developed male abdominal coremata, and 3) lack metepisternal microtymbals (the "Striate Band" of Forbes 1960). With the addition of E. cressida (Dyar), this assemblage corresponds to "Euchaetes Clade 2" of DaCosta et al. (2006). Despite the similarities of Euchaetes nancyae to this group, E. nancyae represents a surprisingly divergent evolutionary lineage within Euchaetes, and should help resolve the phylogeny of this complex group of genera.


s2fieldsofstudy: Biology Medicine

text:
Decreased Expression of CDC25A in Azoospermia as the Etiology of Spermatogenesis Failure.

Background: Spermatogenesis is a tightly regulated developmental process of male germ cells. The stages in spermatogenesis are mitosis, meiosis and spermiogenesis. One of the genes playing a role in meiosis is Cell Division Cycle 25A (CDC25A). Decreased expression of CDC25A is associated with failure of spermatogenesis and sperm retrieval. Infertility examination for azoospermia has been limited on histological examination. Hence, molecular research to find marker genes for infertility will improve the examination of testis biopsies. Methods: This research is a cross sectional study of 50 testicular biopsies with Johnsen scoring categories from scoring 2 to 8. Analysis of mRNA expression used qPCR and protein expression using immunohistochemistry. Statistical analysis with Spearman correlation was considered significant at p<0.05. Results: The result showed that transcript level and protein expression of CDC25A decreased in score 5 of Johnsen scoring categories. Moderate Spearman rho correlation (r=0.546) between mRNA relative expression and protein expression of CDC25A was significant at p<0.01. Conclusion: Decreased expression of CDC25A is associated with meiotic arrest as the etiology of spermatogenic failure in many azoospermic men.

Introduction nfertility is still a problem in reproductive health affecting about 10%-15% of couples (1). Infertility is defined as the inability of married couples to obtain offspring after a year of sexual activity without the use of contraceptives (2). Anatomical abnormalities, endocrine, immunological problems, failure of ejaculation and environmental exposure are factors that may cause infertility (3). Infertility with unknown causes is called idiopathic accounting for about 40% of all infertility cases in men (4). The reason for such high rate of idiopathic infertility is certainly due to the possibility of lack of understanding of the basic mechanisms in the regulation of the genetic control of fertility. About 15%-30% of infertility cases are related to genetic abnormality (5).
The European Guidelines on male infertility recommend Johnsen's scoring for the assessment of the stage of spermatogenesis in the seminiferous tubules (6). Johnsen's scoring comprises 10 categories; a value of 10 denotes complete and regular spermatogenesis with multiple spermatozoa and normal seminiferous tubular epithelium. The scoring categorizes the stages of impaired spermatogenesis gradually down to value of 1 with no seminiferous epithelium.
Disturbances of the process of spermatogenesis involve many genes, approximately 2000 genes are estimated to contribute to the process of spermatozoa formation (7). One important stage in spermatogenesis is the meiotic division that occurs when the primary spermatocyte develops into Suryandari DA, et al. JRI spermatid. Meiosis is the process of cell division through double cleavage and the reduction of the number of chromosomes. The regulation of meiosis at molecular level is not yet fully understood (8).
Several candidate genes have been identified in the regulation of meiosis in animal models. Cessation of spermatogenesis during meiosis (meiotic arrest) is one of the causes of infertility. Meiotic arrest of germinal cells is characterized by being able to enter the stages of meiosis but failing to continue the division process. Hence, seminiferous tubules only contain spermatocytes at the end of the development of germinal cells. Spermatocytes in meiotic arrest will accumulate in the tubules and degenerate. One of the regulator genes of meiotic progression is cell division cycle 25A (CDC25A) (8). CDC25A gene produces a group of proteins that play a role in activating cyclin-dependent kinase (CDK) complex for the regulation of cell division cycles. CDC25A gene is located on chromosome 3 short arm region 21 (3q21) (8). In mammals, there are three isoforms of CDC25, CDC25A, CDC25B, and CDC25C (9). CDC25A isoform is expressed as the most dominant gene in testicles and plays an important role in spermatogenesis (8), particularly in the activation of CDK2-cyclin E and CDK2-cyclin A during the phase of Gap1/ synthesis (G1/S) and possibly also in Gap2/meiotic (G2/M) phase through activation of CDK1cyclin B complex for the initiation of chromosomal condensation. Infertility tests with testicular biopsies have generally been limited to microscopic examination by providing an assessment to the process of spermatogenesis in each seminiferous tubule. Research on genes involved in spermatogenesis failure is important to improve the quality of testicular biopsy examination for the causes of infertility.  (6,10). The biopsies were drawn in the Department of Urology, Dr. Cipto Mangunkusumo Hospital, Faculty of Medicine, Universitas Indonesia, where the informed consent was signed by all participants and immediately transferred to the Department of Medical Biology for further investigation.

Subjects and samples:
Testicular preparation (11): For fixation, testicular tissue was soaked in formalin 10% for 24 hr. Two hours after fixation, the samples were cut crosswise into two parts with a razor blade. To remove water from the tissues, samples were immersed in ethanol 70%, 80%, 95%, and 100%, for 30 min each.
In the clearing process, alcohol was removed from the tissue samples and replaced with a solution of benzyl benzoate for one day, followed by benzyl alcohol for 15 min. For embedding, the clearing agent was removed from the tissue and replaced with liquid paraffin at 60C for about 3 hr to harden. The paraffinated samples were stored at 4-8C in the refrigerator until used for sectioning.
The paraffin blocks were cut by means of a rotating microtome with a thickness of 5 µm in transverse direction. The cross section of the paraffin block was fixed to a glass object slide with Mayer's albumin and then placed on a hot plate.
Deparaffinization and RNA isolation: The detailed procedure of RNA isolation was based on the protocol of High Pure RNA Paraffin Kit (Roche, Germany). In brief, tissue samples from testicular biopsies in the form of paraffin blocks were placed on a bath containing a xylene solution, for 10 min. Then, absolute ethanol was transferred to the bath and incubated for 10 min. The preparation was crushed using a scalpel and moved into Eppendorf cups. The tissue was dried for 10 min at 55C for RNA isolation (12).

JRI
Into the cups, 100 μl tissue lysis buffer was added, and 16 μl of 10% sodium dodecyl sulphate (SDS) and 40 μl of proteinase K working solution were homogenized by Vortex for a few seconds and incubated overnight at 55C. Subsequently, 325 μl of binding buffer and 325 μl of absolute ethanol were admixed to the lysates and centrifuged at a speed of 8000 rotation per min (rpm) for 1 min in a combined high pure filter and collection tube. The obtained supernatant was removed (12).
Then, 500 μl of wash buffer I were added to the tube and centrifuged for 2 min at a speed of 8000 rpm. The supernatant obtained in the bottom collection tube was removed and 500 μl of wash buffer II were added, centrifuged for 2 min at 8000 rpm, and the supernatant obtained in the collection tube was removed. Again, wash buffer II (300 μl) were added, centrifuged for 2 min at 8000 rpm, and the supernatant in the collection tube was removed. The high pure filter tube was centrifuged for 2 min at 12000 rpm for drying and then placed into a new Eppendorf cup. Ninety μl of elution buffer were added and centrifuged for 2 min at a speed of 8000 rpm (12).
Thereafter, 10 μl of DNase incubation buffer and 1 μl of DNase I working solution were admixed to the eluate and incubated for 45 min at 37C. Subsequently, 20 μl of tissue lysis buffer, 18 μl of 10% SDS and 40 μl of proteinase K working solution were added, homogenized by Vortex and incubated for 1 hr at 55C. After incubation, 325 μl of binding buffer and 325 μl of absolute ethanol were admixed into a new combined high pure filter collection tube and centrifuged for 2 min at 8000 rpm. The supernatant obtained in the collection tube was removed and centrifugation repeated at 12000 rpm for drying (12). Now, 500 μl of wash buffer I working solution were added to the reservoir, centrifuged for 2 min at 8000 rpm, and the supernatant obtained in the collection tube was removed. Then, 500 μl of wash buffer II working solution were added and centrifuged for 2 min at 8000 rpm, and the supernatant in the collection tube was removed. The high pure filter tube was centrifuged for 2 min at 12000 rpm, placed into a new 1.5 ml cup; 50 μl elution buffer were added and incubated for 2 min at 20C. After incubation, the cup was centrifuged for 2 min at 8000 rpm to collect RNA, which was subsequently checked for concentration and purity, prior to cDNA synthesis (12). cDNA synthesis: For cDNA synthesis, 10 μl of RNA, 1 μl of oligo (dt) primer, and 2 μl of ddH 2 O were incubated for 10 min at 65°C in a thermal block. Immediately after, the cups were cooled on ice. Into the cups containing the template-primer mix, the remaining components of the RT Mix Transcriptor RT Reaction Buffer, Protector RNase Inhibitor, dNTP and Transcriptor RT were added. Total reaction volume of 20 μl was incubated for 30 min at 55°C and then the transcriptor reverse transcriptase was inactivated by heating to 85°C for 5 min (13).
Glyceraldehyde-3-phosphate dehydrogenase (GA PDH) gene was used as an external standard with forward sequence of 5'-GAAATCCCATCACCA TCTTCCAGG-3' and reverse sequence of 5'-GA GCCCCAGCCTTCTCCATG-3'. The primers were designed to produce a PCR product of 87 base pairs (bp) that would amplify DNA from the base region of 1620 to 1706 (14,15). Quantitative analysis of gene expression was assessed by the method Livak (16). To obtain efficiency value and cycle threshold (Ct), amplification with quantitative real-time (qRT) PCR expression was performed using the SYBR Green I No-Rox kit (Bioline) starting with a singleplex reaction of two replicates (duplo) with a total volume of 20 μl each, in a 96-well optical reaction plate. The qRT-PCR reaction mixture consisted of 5.4 μl of nuclease-free water, 0.8 μl of forward primer, 0.8 μl of reverse primer, 10 μl of SYBR I master mix and 3 μl of cDNA template with a concentration of 100 ng/μl. All qPCR components were incorporated into a 96-well reaction plate with an optical adhesive cover. No template control (NTC) was used to determine the occurrence of contamination during mixing of reagents qRT-PCR. The amplification process was initiated with 1 denaturation cycle at 95°C for 3 min, followed by a cycle at 95°C for 3 s, followed by 40 repeat cycles, annealing and elongation at 56°C for 30 s (16)(17)(18)(19). For qRT-PCR, a 7500 Fast Real-Time Machine (Applied Biosystem, Foster City, United States) was used.
The cDNA synthesis results were further amplified and confirmed by electrophoresis in a 1.5%

Suryandari DA, et al. JRI
agarose gel at 90 volts for 42 min. The band of 87 bp indicated that the samples had good amplification efficiency. Thus, it was possible to continue with primer optimization using qPCR to find the primer attaching to the target gene.

Immunohistochemistry:
Immunohistochemical staining used the TrekAvidin-HRP (horseradish peroxidase) complex labelling with the protocol and kit from Starr Trek Universal HRP-Detection System-Biocare Medical. The primary antibodies against CDC25A were mouse monoclonal antibodies. Positive CDC25A expression was represented by brown staining in the cytoplasm of spermatogenic cells. The microscopic assessment of all expression tests was done on the entire area of small field (magnification 100X) and large field (magnification 400X) (20). The Johnsen scoring system for testicular biopsies categorizes value 10 for complete and regular spermatogenesis with multiple spermatozoa and normal seminiferous tubular epithelium. Category 9 denotes many end-stage spermatids, value 8 less than five spermatozoa and few late-stage spermatids. Value 7 means no spermatozoa, no end-stage spermatids, and many early-stage spermatids, whereas category 6 shows no spermatozoa, no end-stage spermatids, and only few early-stage spermatids. Score 5 denotes no spermatozoa or spermatids and many spermatocytes; score 4 no spermatozoa or spermatids and few spermatocytes. Score 3 shows only spermatogonia; score 2 no germinal cells, only Sertoli cells and in score 1 there is no seminiferous epithelium (6,10). In our study, Johnsen score 8 was used as the control.

Molecular analysis:
RNA was isolated from testicular tissue samples of 50 infertile men with azoospermia at an average amount of 14.81 ng/μl and an average purity value of 2.02. Purity values of 1.9-2.1 indicate that the isolated RNA is pure (23). The subsequent synthesis of cDNA for the gene expression test of CDC25A had an average yield of 1481.13 ng/μl and a purity value of 2.17. Agarose gel electrophoresis showed a band of 87 bp indicating sufficiently good amplification efficiency and primer optimization could be continued using qPCR to determine if the primer binds correctly to the target gene.
The amplification results with the qPCR technique were described in the form of amplification curves. The target gene amplification curve of CDC25A shows the number of cycles with fluorescent signals correlated with the number of target cDNA initiations during the exponential phase of PCR. The amplification curve had a sigmoid shape and showed the value of the Ct indicating the number of cycles captured by the fluorescent signals which correlates to the amplification number (number of copies). The value of Ct varied between 21-29 with an average value of 26.245. In addition, CDC25 gene expression was calculated by comparison with the endogenous control GAP DH (16). indicates an increase in the ratio of relative mRNA expression, but the expression declines in score 5 (0.7414) and then increases again in scores 6 and 7 (1.0974 and 1.0988, respectively), even slightly higher than controls. Statistical evaluation showed abnormal data distribution and significance (p< 0.05) of the correlation between CDC25A mRNA expression and Johnsen scoring.

Percentage of CDC25A positive cells based on
Johnsen categories: Immunohistochemical examination was carried out to determine CDC25A protein expression and visualize localization in spermatogenic cells during spermatogenesis. In table 2, the median percentages of positive cells in different Johnsen assessment categories are given. There is an increase from score 2 to 3 and 4, but in score 5, CDC25A protein expression declines and slightly increases again in score 6. Score 5 of Johnsen categories is characterized by primary spermatocytes that will enter meiosis. In scoring 7 and 8,

JRI
the levels of mRNA expression are considerably higher than in all other groups. A general increase in the values can be seen from score 2 through 8, except for score 5, which decreases versus score 4. Score 6 slightly increases again although its value is still lower than score 4. Statistical evaluation showed strong positive Spearman rho correlation (r s =0.790) between Johnsen scoring and CDC25A-positive cells which was highly significant at p<0.001.

Microphotograph of CDC25A Immunohistochemistry in testicular tissue:
Microphotographs with 400 X magnification show the process of spermatogenesis. CDC25A-positive cells are indicated by brown color of the cytoplasm. Score 2 is characterized by Sertoli cells only, scoring 3 by spermatogonium, scoring 4 is characterized by low amounts of primary spermatocytes, whereas scoring 5 is dominated by primary spermatocytes. Scoring 6 is characterized by the development of spermatogenic cells up to early spermatids and scoring 7 by the development of spermatogenic cells up to late spermatids. Scoring 8 is characterized by complete spermatogenesis from spermatogonium to spermatozoa. The negative control in figure 1H was prepared without primary antibodies, visible are only the blue nuclei of the spermatogenic cells due to counterstaining with hematoxylin Mayer.

Correlation between mRNA relative expression and protein expression:
The correlation test between CDC25A mRNA expression and the number of CDC25A protein-positive cells showed moderate Spearman rho correlation (r S =0.546) with significance at p<0.01. In immunohistochemistry samples, 5 tubules for each sample were used to calculate the number of cells, which was compared with the mRNA expression values according to the type of sample in the immunohistochemical test.

Discussion
This study examined the levels of mRNA and protein CDC25A in testicular tissue of infertile men using qPCR and immunohistochemistry, respectively. It was found that there was a decrease in mRNA expression in patients with spermatogenesis failure of several categories of Johnsen assessment. Score 3 is characterized by spermatogonia cells, where transcription of CDC25A is also found, but with small values.
In Johnson score 4, the CDC25A protein is widely expressed in primary spermatocytes. The decrease in CDC25A mRNA expression indicates an interruption in the spermatogenetic process. In Johnsen score 5, expression values are also lower than the control, probably due to the interruption of meiosis in primary spermatocytes. If the value of the expression is high, the cell division stages will continue. This suggests that CDC25A acts as an important regulator of cell cycle progression or cell proliferation.
The low expression rate of CDC25A in infertile men is the result of the reduction of spermatogenic cycles in the seminiferous tubules and shows that the CDC25 phosphatase contributes to the hyperphosphorylation of M-phase promoting factor (MPF) and CDC25 that are important to complete the meiosis (24). The low CDC25A phosphatase results in inhibition of MPF formation activity that inhibits germinal cells to enter mitosis and meiosis, which results in germ cells having arrest and degeneration occurs (25). The low expression of mRNA is also associated with low levels of protein. This suggests that the  CDC25A content in human testes is regulated at the mRNA level. The upstream factor of CDC25A is probably very important in CDC25A mRNA expression. The human "Deleted in Azoospermia" (DAZ) gene family contains at least three members represented by DAZ, DAZ-like (DAZL) and BOULE, all of its genes being expressed in germ cells. In Drosophila, the expression of Twine protein, which is a Cdc25-type phosphatase, correlates with the intracellular BOULE accumulation and is significantly reduced by BOULE mutants suggesting that BOULE can affect the expression of Twine via a direct bonding to the mRNA twine (26). In mice, Dazl binds directly to 32 UTR of the CDC25A mRNA stating that the DAZ gene family can regulate CDC25A mRNA (26). In addition, CDC25A can accumulate as a result of transcription activation of E2F-1 and c-myc and elevated levels of RNA and protein stimulate cells to enter the cell cycle. Therefore, low CDC25A expression in infertility may be caused by mutations/polymorphisms that affect the family of DAZ, E2F-1 and c-myc genes in the metabolic cascade or other factors that interfere with mRNA production or stability (25). Previous studies of testicular biopsy samples on gene expression analyzed the relationship between the Johnsen assessment with the cAMP responsive element modulator (CREM), protamine 1 and protamine 2 genes and emphasized on stages of spermatogenesis, in particular in the sperm packaging. CREM is a transcription factor of the protamine genes that play a role in the packing of sperm replacing histones. These investigations showed a significant correlation of CREM and protamine with the Johnsen scoring (27).
The presence of protamine in spermatogenesis can cause the buffering of other genes in terms of gene silencing. The expression of protamine mRNA was pronounced in early stages of spermatogenesis resulting in low values of CDC25A gene expression, especially in Johnsen score 5. Low CDC25A expression will result in the disruption of cell division processes leading to spermatogenesis failure at a later stage. In addition, the study by Carrel et al. (8) suggests that protamine can act as a checkpoint for successful spermatogenesis.
Relative expression of the CDC25A gene using real-time quantitative PCR was obtained by amplification of cDNAs with primers designed through Primer3 (14,15). The target gene amplicon of 87 bp was based on adjustment on good primer re-quirements in the amplification of target genes using real-time PCR with an amplicon count of 75-200 bp (19). Although shorter amplicons have higher amplification efficiency, the lower limit of 75 bp makes it easier to distinguish whether a dimer is formed by the primer (19). The primers should have an ideal length of 16-28 nucleotides and a GC content of 35% -65%. The primer used in this study was adjusted to a characteristic length of 20 nucleotides, containing 55% primary and reverse GC with a melting temperature of 56°C; dimer or hairpin structures were not detected (17). The primer optimization for the mRNA expression resulted in melting curves with a single peak indicating primer specificity to the target gene sequence (17,28).
The expression of CDC25A protein was determined by calculating the number of CDC25Apositive cells; this protein is expressed in the cytoplasm. Immunohistochemical methods were performed to determine the location and distribution of proteins in spermatogenic cells in seminiferous tubules (29).
The histopathological examination of the testicular biopsy has a special value in the treatment of cases of male infertility. This assessment is primarily directed against the seminiferous tubules, both in shape and size as well as in the type and number of cells present therein. The use of the scoring method obtained several diagnoses that describe the changes or abnormalities that occur in the testicles (6,30,31).
According to the observations, the CDC25A protein is expressed mainly in spermatocytes. In the histological observation of testicular tissue, secondary spermatocytes are rare or not found because they have little time to enter the next cleavage. In addition to spermatocytes, the CDC25A protein is also found in spermatid cells, sperm cells and is found in spermatogonia and Sertoli cells with a weaker staining intensity than other cells (25). This explains that the CDC25A protein in spermatogonium and Sertoli cells are actively acting cells undergoing mitotic division and CDC25A is a cell cycle regulator of either mitosis or meiosis. Weak staining caused by the CDC25A protein is widely present in the primary spermatocyte cytoplasm resulting in a higher intensity staining of spermatocytes than in spermatogonium and Sertoli cells.
Lin et al. demonstrated that the CDC25A protein was found in the cytoplasm of spermatogonial cells, pachytene spermatocytes stages, early to elongated spermatids from testicular tissue sam-

J Reprod Infertil, Vol 19, No 2, Apr-Jun 2018
Expression of CDC25A in Azoospermia JRI ples (31). In the study by Mizoguchi and Kim, it was mentioned that the location of the CDC25A protein was expressed in the cytoplasm of spermatogonia, spermatocyte, and round spermatid cells, but not found in Sertoli cells and elongated spermatids (32).
The CDC25A protein found in cells that have not been cleaved indicates that the CDC25A function is not only related to cell cycle but also related to physiological process in spermatozoa maturation, and this mechanism is not yet known clearly. Based on the research carried out by Cheng et al., protein CDC25A is found in spermatozoa with immunocytochemical techniques and observed with a confocal microscope andit is known that the tail of spermatozoa shows a stronger color intensity than the head. The tail of the sperm is prepared by mitochondria, outer dense fiber (ODF) and axoneme. The positive results observed in the tail of sperm showed that there is a relationship between the CDC25A protein and the motility of the sperm. The presence of a positive signal of immunostaining results in the head of spermatozoa containing enzymes for the penetration of oocytes shows the role of CDC25A in the fusion of sperm with oocytes. It is also possible that the CDC25A protein is present in the cytosol of the sperm and is not related to specific organelles. The role of the CDC25A protein in motility and the interactions between sperm and oocytes requires further study (25).
The cell cycle transition in eukaryotic cells is regulated by the CDK group. This kinase activity is controlled by other proteins such as cyclin and CDK inhibitors. CDK is inactivated by the inhibition of phosphorylation of Thr-14 and Tyr-15. The inactivated cyclin/CDK complex is dephosphorylated and activated by the CDC25 phosphatase, so CDC25 phosphatase is an important regulator of cell cycle progression (33). In addition, the role of the CDC25A protein in cell division is related to MPF which consists of the catalytic subunit of cyclin B and Cdc. In addition, the role of CDC25A protein in cell division is related to MPF consisting of cyclin B and Cdc catalytic subunit. This MPF required the cells to enter the M phase, either in mitosis or meiosis. This MPF activity is controlled by the dephosphorylation of the CDC25 family and the inhibition of phosphorylation by the Wee-l protein kinase that negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein (34). According to Lin et al., MPF and its regulators are involved in spermatogenesis so that the expression of MPF and its regulators may be correlated with the testicular phenotype and the production of haploid spermatozoa (35).
Research on the relationship between mRNA expression and protein expression is generally carried out because the expression of mRNA depends on the expression of the protein, while the expression of the protein is based on the expression of mRNA. The cellular function depends on the mRNA and protein molecules and the interaction between these two components is important in the management of complex cellular functions (36).
The correlation between mRNA expression and the number of CDC25A-positive cell counts was calculated by the SPSS program. The normality test showed that the normal undistributed data were obtained so that the correlation test was performed. Based on Spearman correlation, p-value of 0.010 (p<0.05) with Spearman correlation coefficient 0.546 was obtained. This suggests that there is a moderate positive correlation between mRNA expression and many positive CDC25A protein cells in the seminiferous tubules. The Spearman correlation coefficient between 0.40-0.70 shows a moderate correlation between two variables (37).
The statistical results show a positive correlation between the relative expression of CDC25A mRNA and the percentage of positive cell counts of CDC25A protein, but when observed in each Johnsen assessment, there is a fluctuating pattern of mRNA expression, a high assessment from 3 to lower assessment 4 and 5. The relative expression returned to increase again in assessments 6 and 7. On the contrary, the pattern of protein expression shows a pattern that increases in value in each expression. Some studies explain that mRNA levels of a gene cannot always predict protein levels. Schwanhausser et al. (38) performed an analysis of mouse fibroblasts and found that abundance of housekeeping gene encoding ribosomal proteins, glycolytic proteins, and Krebs cycle proteins tended to have stability at mRNA and protein levels. In contrast, proteins associated with transcription factors, signal transduction, chromatin modification and cell cycle functions tendency have unstable mRNAs and proteins. The most important regulators in cellular division and differentiation are considered to have a weak correlation between mRNA and protein levels (38).

Suryandari DA, et al. JRI
According to Vogel et al. (39), transcription, mRNA damage, translation, and protein degradation are key processes that determine the stability of protein concentrations. In addition, other factors, like microRNA (miRNA) affect the translation process. It is known that miRNA simultaneously suppresses hundreds of genes by inhibiting the translation of mRNA into protein (40). Post-translational mechanisms also control protein abundance, e.g., hundreds of ubiquitin ligases, proteases, tyrosine and serine-threonine kinases and phosphatases in the human genome that affect the abundance of protein levels; among them, phosphorylases regulate the mechanisms that control cell division and differentiation (41).
Gene expression mostly describes the activity of transcription and does not always correlate with the expression of the protein. This is due to posttranscriptional and post-translational processing that influences the biological protein activity (42). Referring to this study, although the expression of CDC25A mRNA may increase, not necessarily all transcripts are translated and processed to form functional CDC25A protein.

Conclusion
Johnsen scoring 5 exerts highly significant correlation with CDC25A-positive cells indicating CDC 25A protein expression, which in turn correlates with mRNA expression. Decreased CDC25A expression in Johnsen score 5 is associated with meiotic arrest in the etiology of spermatogenic failure. Further studies should clarify how the stimulation and stabilization of CDC25A mRNA and protein expression, e.g., via the influence of protamine can improve spermatogenesis in infertile men.