The Effects of All-Trans Retinoic Acid on Leukocytes in Rat's Embryo
This study was planned to determine the effects of All-Trans Retinoic Acid (ATRA) on the progenitors of White Blood Cells (WBC) and survey their outcomes in rat's embryo during both late-yolk sac and fetal liver stages of hematopoiesis. Single oral dose (100 mg kg-1) of ATRA was administered to rat on Gestation Day (GD) 10 and fetuses were observed on GD 18. The fetus's blood (from experimental group and control each, n = 24) were obtained directly from heart, as placental and mother circulation was continued and subsequently processed for Giemsa staining and followed by WBC counting and measuring. Statistical analysis was made by student t-test. A p-value<0.05 was considered significant. In the experimental embryos on GD 18, the mean number of WBC (29.2%), neutrophil, lymphocyte and monocyte were increased. There was a significant difference in WBC (p<0.0001) and neutrophil (p<0.001) between the groups in this regard. The mean diameter of neutrophil, lymphocyte and monocyte were compared in two groups. The results showed no significant change on experimental and control groups. We concluded that ATRA may have positive effects on proliferation, differentiation and maturation of neutrophil without having any significant effects on the diameter of cells throughout normal granulocyte differentiation in embryo during both late-yolk sac and fetal liver stages of hematopoiesis.
The commitment of embryonic cells to hematopoietic fates begins in proximal
region of egg cylinder at the mid-primitive streak stage with the simultaneous
appearance of primitive erythroid and macrophage progenitors (Palis
et al., 1999). First, the differentiation of leukocytes occurs and
followed by erythrocytes differentiation (Tashiro et
al., 2006). The first leukocytes that arise in the development of vertebrate
embryos are the primitive macrophages, which differentiate in the yolk sac and
then, quickly invade embryonic tissues (Le Guyader et
al., 2008). It is apparent that a wide variety of external and internal
stimuli influence and modulate the lineage choice and differentiation during
hematopoiesis (Bedi and Sharkis, 1995). Hematopoiesis
is a highly regulated process resulting in the formation of all blood lineages
(Buitenhuis et al., 2008).
Retinoids are a group of natural and synthetic vitamin A analogues and exert
important effects on the growth and differentiation of various cell types including
hematopoietic progenitors (Douer and Koeffler, 1982).
Also, there is a strong body of evidence that vitamin A (Kang
et al., 2007) and retinoids have immune regulatory functions (Kang
et al., 2007; Chen et al., 2009) and
improve the numbers of various immune cell types (T helper 2) (Iwata
et al., 2003). The fact that Retinoic Acid (RA) is a differentiating
agent for granulocyte in culture has been well established for both normal and
leukemic cells (Lawson and Berliner, 1999). However,
the effects of exogenesis All-Trans Retinoic Acid (ATRA) throughout normal granulocyte
differentiation during both late-yolk sac and fetal liver stages of hematopoiesis
have so far not been investigated. Therefore, in this study, we have examined
the effects of ATRA on the progenitors of White Blood Cells (WBC) and survey
MATERIALS AND METHODS
Animals: In this study, Wistar strain of rats (about 3 months of age) were used. They were housed in light (12:12 light: dark cycle) and temperature-controlled (21°C) rooms and maintained on laboratory chow and tap water provided ad libitum. Adult virgin females (n = 10) (180-200 g in weight) were mated overnight with males of the same stock. A vaginal plug and smear observed indicated day 0 of pregnancy. The experimental protocol was approved by the Ethics Review Committee for Animal Experimental of the Semnan University of Medical Sciences. This study was conducted from 2007 to 2009.
Drug: ATRA (Sigma-Aldrich, USA) were used in this study.
Preparation of solution: Single dose of 100 mg kg-1 of ATRA
(Sigma-Aldrich, USA) suspended in alcohol; corn oil (1:9) mixture (Padmanabhan,
1998) (light-proof containers under yellow light) was administered. The
drug was given by oral incubations on the morning of Gestation Day (GD), 10
to experimental group (n = 5). The control (n = 5) were vehicle treated. The
animals were euthanized on GD 18 by ether.
Samples preparation: The fetus's blood (experimental and control each,
n = 24) were obtained directly from heart, as placental and mother circulation
was continued. Blood cytology slides were made using the cytospin preparation
(Akbari et al., 2007) and subsequently processed
for staining and followed by counting and measuring. These measurements in each
sample were done in ten slides and in the five areas for each section (using
Randomization Implementation). The embryos weighed and fixed in 10% formalin,
examined for external malformation. The blood cells were studied by light microscope
and eye piece (x200 for WBC count and x1000 for diameter).
Statistical analysis: The values were expressed as Mean±SEM.
Comparison between two groups was made by student t-test (Mohammadi
et al., 2009), p<0.05 was considered as significant different.
The experimental group showed a marked malformation on external inspection.
In the experimental embryos on GD 18, the number of WBC per field was 42.12±2.14
with 29.3% increase in this group, compared to control group (32.58±0.88)
as was shown in Fig. 1. In the experimental group, the number
of neutrophil per field was 12.8±1.50 with 85% increase in this group,
compared to control group (6.9±0.30) as was shown in Fig.
1. In the experimental embryos, the number of lymphocyte per field was 20.6±1.32
with 16.4% increase as compared to control group (17.75±1.15), no significant
difference was found between two groups. The number of monocyte per field in
experimental group was 8.6±0.56 with 8.4% increase when compared to control
group (7.91±0.24).There were significant relationships between two groups
in WBC (p<0.0001) and neutrophil (p<0.001) counts. However, there were
no significant changes in monocyte and lymphocyte numbers in two groups Fig.
1. The mean diameter of neutrophil, lymphocyte, monocyte, for experimental
and control groups were: 6.88±0.18 and 7.26±0.13; 5.8±0.13
and 5.6±0.15; 8.35±0.24 and 8.18± 0.18, respectively (Fig.
Effect of ATRA (100 mg kg-1) on WBC, neutrophil,
lymphocyte and monocyte numbers of rat embryo on GD 18. *p< 0.0001,
**p< 0.001 relative to values in experimental group. Results was presented
as Mean±SEM for n = 24 embryo per group
of ATRA (100 mg kg-1) on the mean diameter of WBC in rat embryo
on GD 18, in comparison with control group. No significant differences
between groups were noted in this regard
These data showed that the mean diameters of leukocytes were not markedly
ATRA and diverse synthetic analogues affect differentiation of neoplastic and
normal hematopoietic cells (Lanotte et al., 1991).
In this study, a single dose of ATRA has been used to identify its effects during
hematopoietic differentiation in rat's embryos. The data indicated that ATRA
enhances the proliferation and differentiation of myeloid compartment, especially
The increase in the neutrophils population, was seen here, shows that exogenous
ATRA is able to improve the proliferation and differentiation of their progenitors
during both late -yolk sac and fetal liver stages of hematopoiesis in embryo.
These results are in line with those of Leelasiri et
al. (2005), which reported an increase in neutrophil number after ATRA
treatment, as well as with a reports showing induced granulocytic differentiation
of acute myeloblastic, promyelocytic leukemia cell lines (Valtieri
et al., 1991) and acute promyelocytic leukemia blasts in vivo
(Warrell et al., 1991). In addition, this finding
is consistent with results from previous studies showing that ATRA enhances
the Hematopoietic Progenitor Cells (HPCs) in vitro (Collins
et al., 2001; Tobler et al., 1986).
In the hematopietic system, RARα is expressed at significant level in granulocyte
(Labrecque et al., 1998) and stimulates differentiation
in response to exogenous RA. This receptor can bidirectionally modulate granulopoiesis
as a differentiation factor when liganded to RA or as an inhibitor in the absence
of ligand (Kastner et al., 2001). Our data about
lymphocyte and monocyte were in line with the results of Seguin-Devaux
et al. (2005), who determined that RA directly enhances the number
of T lymphocytes in the peripheral blood. Although these enhancing effects were
not significant. It was contradicted with previously reports showing that RA
decreases proliferation of lymphocyte (Stosic-Grujicic and
Ejdus, 1994) and human myeloid leukemia cells in vitro (Douer
and Koeffler, 1982).
Macrophage progenitors (Mac-CFC) increase in numbers in the yolk sac until
GD 9.5 and are preferentially localized to the tail and para aortic splanchopleura
aorta-gonad-mesonephros region prior to the development of the liver. These
progenitors increase within the fetal liver between GD10.5 to 11.5. GM-CFC could
also be detected within the yolk sac on GD 8.5. Low numbers of them persisted
in both the yolk sac and embryo proper until GD 11.5, at which time that significant
numbers were present in both blood and fetal liver. These progenitors gave rise
to small to medium size colonies that contain cells with both neutrophils and
macrophage morphology (Palis et al., 1999). It
should be noted that when ATRA was given, the progenitors of leukocyte, certainly
were Mac-CFC and bipotential granulocyte/macrophage (GM-CFC) in the embryo.
The apparent difference in the responses of neutrophil, lymphocyte and monocyte
to ATRA may be as under: It is hypothesized that the inhibitory effects of RA
on erythroid, monocyte and high-proliferative potential colony-forming cells
originated through activation of either RAR/RXR of D3R/D3R-D3R/RXR, while, the
stimulatory activity on granulocytic colony formation could theoretically be
described only to activation of RAR/RXR (Tocci et al.,
1996). The exogenous RA is able to redirect erythroid-monocyte, or eosinophil-committed
progenitors toward a granulocyte fate (Tobler et al.,
1986; Tocci et al., 1996; Paul
et al., 1995). In addition, it possibly induces an HPC shift from
erythroid to granulocytic neutrophilic-differentiation pathway (Labbaye
et al., 1994). It also appears to orient the differentiation of pluripotent
hematopietic progenitors toward the granulocyte lineage (Tocci
et al., 1996). Thus, it may be involved as a commitment factor (Paul
et al., 1995) and fate specification for this lineage (Kastner
et al., 2001; Seguin-Devaux et al., 2005)
with different effective concentrations in vitro and in vivo.
After data analysis, we found that ATRA not to be of significant influence
on cell diameter. These finding are in contrast with those of Kinoshita
et al. (2000), who reported a decrease in mast cell's diameter with
this agent. The size of a cell depends on intrinsic and extrinsic factors and
is regulated by Ribosome biogenesis in multicellular organisms (Hafen
and Stocker, 2003). That is also a consequence of correlation between the
rates of cell-cycle progression and growth, but four different general processes
modulate the two rates (Rupes, 2002). Although, the
mechanisms responsible for the differences in cell size among different red
cell lineages are not well understood, they may relate to differential rates
of cell division (Kingsley et al., 2004). This
issue needs future investigations.
This study shows that administration of exogenous ATRA during both late-yolk sac and fetal liver stages of hematopoiesis may be improve proliferation, differentiation and maturation of neutrophils, without having any deleterious effects on the dimenation of WBC.
This study was supported by grant from Semnan University of Medical Sciences and Health Services.
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