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Introduction: For differentiation and maintenance of epithelial cells in vitro and vivo under the influence of retinoic acid treatment skin seems to be a major target organ for both the normal and pathological states. The current study was conducted to evaluate the effects of retinoic acid on fetal skin if given to albino mice during pregnancy.Materials and Methods: Twelve pregnant albino mice were divided into two groups of 6 each; the experimental group was given 60 mg/kg/day of retinoic acid (R.A) dissolved in 0.1ml of olive oil orally on 78 and 9 day of gestation. Fetuses were delivered and dissected on 18th day of gestation; skin samples were removed and processed for microscopic study.Results and Conclusion: Histological examination of fetal skin in RA treated group showed increased keratinocyte proliferation resulting in increased number of epidermal cell layers and increased epidermal thickness. It also inhibited the development of hair follicles which are seen in the form of rudimentary buds in the dermis. So its usage during pregnancy should be warranted. Given the essential role of retinoids in epidermal differentiation and their effectiveness in the treatment of several skin orders it is important to see its histological effects on skin.

Key words: Retinoic acid skin dams fetus proliferation.


Retinoids are naturally occurring and synthetic analogues of vitamin A which is essential for embryogenesis growth and epithelial differentiation.1Maternal vitamin A deficiency or retinoic acid (RA) excess results in a spectrum of congenital malformations in a dose and developmental stage dependentmanner.23 Thus RA levels must be strictly controlled and should be consumed with caution during pregnancy.The effects of RA are mediated by two major groups of peptides the nuclear receptor proteins and the cytoplasmic binding proteins. There are two members of retinoic acid receptors: retinoic acid receptors (RAR) and retinoic X receptors (RXR).4Each receptor family has at least three subtypes (a AY and ). Another study demonstrated expression of RAR gamma in human epidermis and suggested that RAR gamma is a molecular target of RA action in adult human skin.5-7RA RAR complexes bind to short cis acting DNA sequences hormone responsive elements near the promoters of target genes in order to regulate the target gene expression.8Morphogenesis of embryonic tissue is greatly affected by retinoids. The pattern of mouse vibrissae follicles are modified to form mucus gland pattern and alsofeather epidermis of the chick are reported to be transformed from the chick scales epidermis.9In mammalian hair follicles development there is reciprocal induction between ectodermal epithelium and mesodermal mesenchyme. In mouse it begins at14th day of gestation by local proliferation of epidermis forming thickenings (placode stage) followed by hair germ stage then hair peg stage (dermal papilla formation) and finally hair follicles are formed.10For chemoprevention or treatment of skin disorders including malignant conditions retinoids are found to be effective agents. Major role in growth and differentiation for a variety of malignant and normal cells retinoids are regarded to have an important function.They are known to play a major role in regulatinggrowth and differentiation of a variety of normal and malignant cells.11Topical all trans RA treatment caused an increase in the number of living cell layers in the epidermis of both human and mouse.1213It has been reported that retinoic acid treatmentresults in expression of post-transcriptional elevation in epidermal growth factor receptors (EGF R).14The epidermal growth factor receptor (EGFR) is the cell surface receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. Upon activation by their ligands there is stimulation of tyrosine kinase pathways that ultimately initiate several signaling pathways involving cellular proliferation differentiation and apoptosis.1516RA alters expression of epithelial growth factor receptor (EGFR) signaling pathways as in kidney epithelial and fetal lung cell lines; it was also found to modulate the action of EGF in developing skin of mouse embryos.17 On this basis we have tried to see the histological effects of administration of retinoic acid to pregnant dams on developing skin of mouse fetuses to substantiate the earlier observations.


Sixteen albino mice (twelve female and four males) weighing 25 30 gm and 6 8 weeks old were procured from National Institute of Health Islamabad. Animals were housed in the Research laboratory of University of Health Sciences Lahore under controlled conditions of temperature (22 0.5C) humidity (50 10%) and light and dark cycle of 12 hours each. They were given mouse chow and water ad libitum.The experimental animals were randomly divided into two groups of eight animals each six female and two males. Female mice were caged overnight with normal males in the ratio of 1male: 3 females. Mating was confirmed by the presence of a vaginal plug on the following morning and was considered gestational day0 (zero).18Animals of control group were given 0.1ml of olive oil orally on 7th 8th and 9th day of pregnancy whereas experimental group received 60 mg/kg/day of retinoic acid dissolved in 0.1ml of olive oil orally on comparable days of pregnancy. Pregnant mice were sacrificed and dissected on the 18th day of gestation to obtain the fetuses. The fetal skin samples were taken from limb and dorsal aspect of trunk for histological preparation; these were fixed in 10% formalin for 48 hours and later processed for preparation of paraffin blocks. Sections5 thick were obtained using Leica rotatory microtome (RM 2125); stained with Haematoxylin and Eosin for light microscopic examination.


The thickness of epidermis was measured using ocular micrometer at X40 objective after calibrating it with linear stage micrometer. The number of hair follicles per mm was counted under 40X magnification using an ocular reticule. The hair follicles were counted at four sites and then their mean was taken.

Statistical Analysis

The statistical analysis was carried out using computer software Statistical package for social sciences (SPSS) version 16. The arithmetic mean standard deviation and the significance between two groups was calculated by Mann-Whitney test. The difference was regar-ded statistically significant if the p' value was = 0.05.


At gestational day18 histological examination of fetal skin from the control group revealed a well organized epidermis with distinct layers of closely packed cells. It was characterized with prominent stratum basal and corneum (Fig. 1).In treated group epidermal layers were not evidently discernible and there appeared to be gaps betweenthe cells which were presumably due to accumulation of inter cellular matrix. Epidermis was discernibly thicker than that of the control group; this was possibly due to an increase in the number of cells resulting from rapid proliferation of those in stratum basal (Fig. 2). There was no change in dermal connective tissue of the skin of both groups. Mean epidermis thickness of treated group was higher when compared to that of the control groups the difference was statistically significant (p-valueless than 0.001).In addition hair follicle morphogenesis was also altered in the treated group; they were reduced in number and their growth was arrested at germ stage of hair follicle development. They did not invaginate deeply into dermis and rudimentary buds were found.The mean hair follicle count of treated group was lower than that of the control and the difference was statistically significant p-value less than 0.001.


Growth and differentiation of epithelial tissues are greatly affected by retinoids. Changes in keratinocyte morphology when treated with different doses of various retinoids in earlier cell culture studies had been reported. Retinoic acid receptor-gamma (RAR gamma) is one of the variety of retinoic acid receptors and is reported to be present exclusively in skin.19 RAR-a and RAR- play a role in epidermal dermal interactions that lead to hair follicle morphogenesis.20In present study pregnant female mice were given60 mg/kg/day of retinoic acid dissolved in 0.1 ml of olive oil orally on 7th 8th and 9th day of pregnancy; significant concentrations of all trans RA in the plasma and embryo were reached after oral treatment of pregnant dams in mouse rat and rabbit; administration of RA in high concentration had in the past been reported to produce teratogenic effects on account of its metabolites filtering through placental barrier.2122Histological structure of fetal skin in the controlgroup showed stratified epidermis with distinct layers of closely packed cells whereas fetal skin from the treated group showed increased thickness of epidermis due to increased proliferation of cells in stratum basal induced by RA. Similar findings were reported earlier in which single topical application of all-trans retinoic acid for 4 days stimulated keratinocyte proliferation increasing the number of epidermal cell layers and increasing epidermal thickness.23Another study demonstrated aromatic retinoid induced epidermal hyper-proliferation and numericalhyperplasia after oral administration of the drug in the animal model of the hairless mouse.24Increasing EGF binding capacity in several cell types after RA treatment also has been found.25 Mechanism of action of epidermal growth factor (EGF) is pre-

Table 1: Showing comparison between parameters of fetal skin when compared among groups.

###Control Group Treated Group

###Parameter###(n = 45)###(n = 42)###p-value

###Mean ( S.D)###Mean ( S.D)


###131.20 24.50 102.09 25.95###0.001

thickness (m)

No of hair

###38.72 5.93###61.07 12.49 0.001

follicles / mm2

sumed to occur through binding with EGFR resulting in anincrease in the tyrosine kinase activity of receptor. It subsequently triggers a cascade of intracellular events leading to increased DNA synthesis and cell proliferation.26In another study it was found that RA activated the transcription of two different nuclear receptors RAR and PPARAY/d; the activation of PPARAY/d lead to expression of genes producing cell proliferation.27Fetal skin in treated group also showed that layers of the epidermis were not characteristically structured and the large gaps between the cells were observed. These findings are consistent with another study in which RA lead to accumulation of Hyaluronate (HA) producing gaps between the cells in the superficial layers of epidermis; this was produced by increased activity of keratinocytes.28Hair follicle morphogenesis and hair cycling are controlled by complex bidirectional ectodermal mesenchymal interactions between epidermal keratinocytes and a specialized population of fibroblasts with inductive morphogenic properties which mature into dermal papilla of the hair follicle.29 In our study number of hair follicles were reduced in treated group and most of them were arrested at germ stage. Similar findings were reported earlier by Okano et al (2012) in which increased RA levels reduced the density of hair follicles and arrested hair follicle growth: further in retinoic acid treated dams in mouse the fetuses were ablated on account of degradation of enzyme Cyp26b1 (cytochrome P450 family 26 subfamily b polypeptide1).30Vitamin A treated cultures altered differentiation and increased proliferation of human keratinocytes as reported by Chopra;31 however this did not offer an explanation to its therapeutic effect seen in various skin diseases of hyperkeratosis. Additional work is suggested in this direction.


Facilities and financial support provided by the Uni-versity of Health Sciences Lahore and the help by the technical staff is greatly appreciated.


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Date:Mar 31, 2014

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