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Fulltext Cardiovascular and metabolic responses to isometric exercise of athletes and non-athletes

Rajikin MH

Med J Malaysia, 1980 Sep;35(1):46-52.
PMID: 6973059

Seven non-anthletes [21-26 yrs] and five athletes [23-29 yrs] have participated in the study of cardiovascular and metabolic responses to 15 minutes isometric leg exercise at 15% of each of their maximal voluntary contraction force [MVC]. Oxygen consumption [V02 ]. heart rate [HR] and blood pressure (BP]. were measured during resting. exercise and recovery! periods. The results show that there were a significant increase in V02. HR and BP of the two groups studied during exercise, but no significant difference between groups has been found. This study indicates that although cardiovascular response between the rwo groups was not significantly different, 15 minutes isometric leg exercise at 15% MVC has challenged this system in a such a way to fulfill the metabolic requirement of the
working muscle ill both groups. This finding was
discussed in light of the available literatures.
Fulltext Variations of serum prolactin levels in Malay women from premenarche to the postmenopause

Rajikin MH, Satgunasingam N

Med J Malaysia, 1984 Jun;39(2):135-8.
PMID: 6513852

A double-antibody radioimmunoassay technique has been used to investigate the serum prolactin (hPRL) level in Malay females from premenarche to the postmenopause. The results showed that the hPRL level (mena ± SEM) in the premenarchal, postmenarchal and late pubertal/reproductive subjects were 23.6 ± 2.3, 19.1 ± 2.0 and 22.7 ± 1.9 ng/ml respectively. In premenopausal women, hPRL level (11.8 ± 2.4 ng/ml) was significantly reduced (p < 0.01) compared to that of late pubertal group; the level declined even further after menopause (9.5 ± 1.7 ng/ml). Although the difference in the mean prolactin levels between premenopause and postmenopause were not significant, 73% of the postmenopausal women had serum prolactin concentrations below 10 ng/ml compared to 44% of the premenopausal and 10% in late pubertal group.
Fulltext Transfer factor in smokers and non-smokers after smoking two cigarettes

Rajikin MH, Etta KM

Med J Malaysia, 1989 Mar;44(1):23-9.
PMID: 2626109

Impairments of lung functionality as long-term effects of cigarette smoking have been well established. To our knowledge, no study on acute recovery patterns in any important lung function index after smoking a very limited number of cigarettes has ever been reported. The present study reports recovery patterns of lung transfer factor (TF) and related parameters in smokers and non-smokers who smoked two Camel cigarettes. Lung transfer factor and other indices were determined by the single breath-holding technique. From our results, the TF and related indices of healthy Malaysians are similar to previously published normals of comparable age. On smoking two cigarettes, male smokers began to recover from the 30th minute; male non-smokers had not begun recovery even by the 50th minute. Extrapolation of the recovery curves suggests that a "safer" interval between cigarettes for male smokers is about 114 minutes.
Fulltext The disappearance of sleep-related changes of serum prolactin in pregnant women

Rajikin MH, Abdullah R, Hamid Arshat

Med J Malaysia, 1983 Dec;38(4):311-4.
PMID: 6599989

Serum prolactin (hPRL) levels in nonpregnant, pregnant and postpartum women during sleep were investigated. The study showed that in non-pregnant women, there is an immediate shift of hPRL release with reversal of sleeping period. Thus, the nocturnal surge for prolactin is sleep related. In pregnant women, however, while there is an increase in hPRL level during pregnancy, the nocturnal rise of this hormone is not detected and this is observed as early as the first trimester of pregnancy.
Fulltext Serum prolactin levels during pregnancy, intrapartum and postpartum periods in Malay women

Rajikin MH, Abdullah R, Arshat H, Satgunasingam N

Med J Malaysia, 1982 Mar;37(1):72-5.
PMID: 6981751

Serum human prolactin (hPRL) levels in Malay women during pregnancy, intrapartum and immediately postpartum have been invest£gated by means of a double-antibody radioimmunoassay technique. There was a progressioe rise of serum prolactin concentration from 31.9 ± 10.4 ng/ml in the first trimester to 242.0 ± 24.6 ng/ml at 36 weeks pregnancy unth. the mean values during the second and third trimester of 118.9 ± 12.7 ng/ml and 214.7 ± 10.3 ng/ml respectively. During intrapartum the concentration of hPRL was 191.9 ± 26.9 ng/ml and ithat of immediately postpartum was 178.3 ± 14.5 ng/ml. Suckling of the breast within 6 hours postpartum has resulted in a minor elevation of prolactin level, and this could have been due to the greater basal prolactin level, and/or the ineffectiveness of suckling as a potent stimulus during this period.
Fulltext Serum prolactin levels at different stages of menstrual cycle and during a 24-hour period in Malay women

Rajikin MH, Abdullah R, Arshat H

Med J Malaysia, 1981 Sep;36(3):155-8.
PMID: 7199111

Radioimmunoassay technique has been used to investigate the variation in the serum human prolactin (hPRL) levels at different stages of menstrual cycle and during a 24-hours period in Malay women. The results showed that the serum hPRL concentrations during menstrual (24.5 ± 4.3 ng/ml), preovulatory (36.6 ± 7.4 ng/ml), ovulatory (29 ± 5.3 ng/ml) and postovulatory (26.6 ± 5.2 ng/ml) periods were not significantly different among each other. The hPRL level was highest during night-sleeping, at 0200hr and was significantly different from the values obtained at 1400, 2000, 0800 and 1400 (last sampling) hours (P < 0.01).
Ultrastructural studies of developing goat oocytes in vitro

Rajikin MH, Yusoff M, Abdullah RB

Theriogenology, 1994 Nov 01;42(6):1003-16.
PMID: 16727604

The structure and distribution of organelles within developing goat oocytes at various stages of incubation were studied. In oocytes with 5 or more layers of cumulus cells, at 0 h of incubation, the zona pellucida had developed although zonation was not evident. Lipid bodies were present but no mitochondria were observed. At 20 h, the zona pellucida had differentiated into thicker and thinner regions. Clusters of membrane-bound electron-transparent bodies were present in the perivitelline space. The mitochondria were fully developed, distributed evenly and usually in close proximity with dilated endoplasmic reticula. Cortical granules were distributed at the periphery. At 40 h of incubation, a number of mitochondria was hooded. In oocytes of 2 to 4 layers of cumulus cells at 0 h, the zona pellucida was penetrated by cumulus cell processes, and the mitochondria were not well developed. However, in 20-h incubated oocytes, fully developed mitochondria, many of which were hooded, could be observed. Clusters of membrane-bound electron-transparent bodies were also observed, while cortical granules were at the periphery. In cumulus-free oocytes, zonation within the zona pellucida was indistinct. Very few vesicles and lipid bodies were observed. At 20 h, mitochondria were sparsely distributed and were not well developed and lacked cristae. At 40 h, the zona pellucida was less compact, and the membrane-bound electron-transparent bodies were less numerous compared with those of the other groups. Endoplasmic reticula were not dilated, and cortical granules were few and had no definite pattern of distribution.
Diabetes alters the mRNA expression of insulin-like growth factors and their receptors in the mouse fallopian tube and uterus during the preimplantation stages

Zakaria R, Rajikin MH, Yaacob NS, Nor NM

Reprod Biol, 2007 Mar;7(1):41-53.
PMID: 17435832

The possible role of insulin-like growth factors (IGFs) and their receptors (IGFRs) in the pathogenesis of diabetic embryopathy was investigated. Sexually mature female ICR mice of 6-8 weeks old were made diabetic by a single intraperitoneal injection with 200 mg/kg streptozotocin ten days prior to mating. Fallopian tubes and uterine tissues were obtained from the superovulated diabetic and normal mice 48, 72 and 96 hours following human chorionic gonadotropin (hCG) injection. The mRNA expression of IGF-1 and IGF-2 as well as their receptors was determined in the tissues using Real-time Polymerase Chain Reaction (Real-time PCR). The mRNA expression of IGF-1 in the fallopian tube and uterus of the diabetic mice was significantly lower 72 and 96 hours after hCG treatment, respectively, as compared to the controls. The mRNA expression of IGF-1R at 96 hours post-hCG treatment was significantly higher in the fallopian tube and lower in the uterus of the diabetic mice as compared to the controls. The mRNA expression IGF-2 in the fallopian tube was significantly higher 48 and 96 hours after hCG treatment, but was lower in the uterus of diabetic mice 96 hours after hCG treatment as compared to controls. The mRNA expression of IGF-2R in the diabetic mice was significantly higher 48 and 96 hours (the fallopian tube) and 48 hours (uterus) after hCG treatments as compared to the controls. In conclusion, an alteration in mRNA expression of IGFs and their receptors in the diabetic mice as observed in this study could possibly result in diabetic embryopathy.
The effects of thyroxine treatment on slow- and fast-contracting skeletal muscle contractions of the cat and their cyclic AMP level

Merican Z, Sukumaran S, Raji VL, Rajikin MH, Khalid BA

Clin Exp Pharmacol Physiol, 1992 Dec;19(12):843-6.
PMID: 1335382

1. The effects of thyroxine treatment on soleus and extensor digitorum longus (EDL) muscle contractions and their cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels were examined in anaesthetized cats. 2. Thyroxine treatment decreased the tension of incomplete tetanic contractions of the soleus as well as the EDL muscles. The effect on tension of these muscles was not associated with an increase in the cyclic AMP level of the muscle as is the case with a beta 2-adrenoceptor agonist effect. 3. The results do not support the involvement of cyclic AMP in the tension depressant effect of thyroxine on contractions of skeletal muscle. 4. It is suggested that the muscle weakness and tremor observed in thyrotoxicosis and during administration of beta 2-adrenoceptor agonists are mediated by different mechanisms.
Immunolocalization of insulin-like growth factors and their receptors in the diabetic mouse oviduct and uterine tissues during the preimplantation period

Zakaria R, Rajikin MH, Yaacob NS, Nor NM

Acta Histochem, 2009;111(1):52-60.
PMID: 18676006 DOI: 10.1016/j.acthis.2008.04.002

The aim of the present study was to analyze the immunolocalization of insulin-like growth factor (IGF)-1 and IGF-2 and their receptors in the oviduct and uterus of control and diabetic mice. Sexually mature female ICR mice aged 6-8 weeks were rendered diabetic by streptozotocin (200 mg/kg, administered intraperitoneally). Oviductal and uterine tissues were obtained from the superovulated control and diabetic mice at 48, 72 and 96 h post-human chorionic gonadotropin (hCG) treatment. Localization of IGF-1, IGF-2, IGF-1R and IGF-2R was determined by immunohistochemistry and a semi-quantitative scoring of immunolabelling was performed using a standardized 5-point system. The immunohistochemical scorings for both IGF-1 and IGF-1R were significantly decreased in the oviducts of diabetic mice at 96 h post-hCG treatment. The scores for IGF-2 were significantly increased in the oviducts of diabetic mice at 48 and 72 h post-hCG treatment, and for IGF-2R at 72 h post-hCG treatment. However, there was no significant difference in the scores of IGFs and their receptors in the uterus of control and diabetic mice. In conclusion, the oviductal immunolabelling for IGFs and their receptors was significantly altered by maternal diabetes, which may be of importance in the pathogenesis of preimplantation diabetic embryopathy.
Nicotine and endometrial decidual growth in pseudopregnant rats

Siti Norashikin MT, Ghosh S, Chatterjee R, Rajikin MH, Chatterjee A

Reprod. Med. Biol., 2014 Jul;13(3):135-141.
PMID: 29699157 DOI: 10.1007/s12522-013-0174-9

Purpose: The present study aims to investigate the effects of nicotine on the endometrial decidual growth and levels of estrogen and progesterone in pseudopregnant rats.
Methods: Pseudopregnancy (pc) was induced in cyclic Sprague-Dawley rats by sterile mating. Subcutaneous injection of nicotine tartrate (7.5 mg/kg/day) was scheduled from day 1 through day 5, day 5 through day 9 or day 1 through day 9 of pc. In another group of pseudopregnant rats, concomitant treatment of nicotine tartrate concurrently with progesterone (2 mg/day) was scheduled from day 1 through day 9 pc. Control groups received subcutaneous injections of vehicle only. Endometrial decidualization was induced on day 5 pc. On day 10 pc, animals were sacrificed.The degree of decidual growth and circulating levels of estrogen and progesterone were measured.
Results: The decidual growth in all the first three nicotine-treated groups of animals was significantly reduced, particularly in the animals treated with nicotine from day 1 through day 9 pc. Plasma estrogen levels were significantly elevated in animals treated with nicotine from day 1 through day 9 pc. Conversely, levels of plasma progesterone were found to be significantly attenuated in the same group of nicotine-treated animals compared to controls. Exogenous replacement of progesterone, however, caused a higher degree of endometrial decidualization compared to the nicotine-treated group but it was slightly less than when compared to control.
Conclusions: In conclusion, nicotine-induced progesterone deficiency with a corresponding elevation of estrogen may possibly attenuate the degree of endometrial decidualization in pseudopregnant rats.
Annatto ( Bixa orellana ) δ-TCT supplementation protected against embryonic DNA damages through alterations in PI3K/ Akt-Cyclin D1 pathway

Mutalip SSM, Rajikin MH, Rahim SA, Khan NMN

Int J Vitam Nutr Res, 2018 Feb;88(1-2):16-26.
PMID: 30907699 DOI: 10.1024/0300-9831/a000492

Protective action by annatto-derived delta-tocotrienol (δ-TCT) and soy-derived alpha-tocopherol (α-TOC) through the regulation of PI3K/Akt-Cyclin D1 pathway against the nicotine-induced DNA damages is the focus of the present study. Nicotine, which has been widely reported to have numerous adverse effects on the reproductive system, was used as reproductive toxicant. 48 female balb/c mice (6-8 weeks) (23-25 g) were randomly divided into 8 groups (G1-G8; n = 6) and treated with either nicotine or/and annatto δ-TCT/soy α-TOC for 7 consecutive days. On Day 8, the females were superovulated and mated before euthanized for embryo collection (46 hours post-coitum). Fifty 2-cell embryos from each group were used in gene expression analysis using Affymetrix QuantiGene Plex2.0 assay. Findings indicated that nicotine (G2) significantly decreased (p < 0.05) the number of produced 2-cell embryos compared to control (G1). Intervention with mixed annatto δ-TCT (G3) and pure annatto δ-TCT (G4) significantly increased the number of produced 2-cell embryos by 127 % and 79 % respectively compared to G2, but these were lower than G1. Concurrent treatment with soy α-TOC (G5) decreased embryo production by 7 %. Supplementations with δ-TCT and α-TOC alone (G6-G8) significantly increased (p < 0.05) the number of produced 2-cell embryos by 50 %, 36 % and 41 % respectively, compared to control (G1). These results were found to be associated with the alterations in the PI3K/Akt-Cyclin D1 gene expressions, indicating the inhibitory effects of annatto δ-TCT and soy α-TOC against the nicotinic embryonic damages. To our knowledge, this is the first attempt on studying the benefits of annatto δ-TCT on murine preimplantation 2-cell embryos.
Fulltext Vitamin E as an Antioxidant in Female Reproductive Health

Mohd Mutalip SS, Ab-Rahim S, Rajikin MH

Antioxidants (Basel), 2018 Jan 26;7(2).
PMID: 29373543 DOI: 10.3390/antiox7020022

Vitamin E was first discovered in 1922 as a substance necessary for reproduction. Following this discovery, vitamin E was extensively studied, and it has become widely known as a powerful lipid-soluble antioxidant. There has been increasing interest in the role of vitamin E as an antioxidant, as it has been discovered to lower body cholesterol levels and act as an anticancer agent. Numerous studies have reported that vitamin E exhibits anti-proliferative, anti-survival, pro-apoptotic, and anti-angiogenic effects in cancer, as well as anti-inflammatory activities. There are various reports on the benefits of vitamin E on health in general. However, despite it being initially discovered as a vitamin necessary for reproduction, to date, studies relating to its effects in this area are lacking. Hence, this paper was written with the intention of providing a review of the known roles of vitamin E as an antioxidant in female reproductive health.
Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo

Jusof WH, Khan NA, Rajikin MH, Satar NA, Mustafa MF, Jusoh N, et al.

Int J Fertil Steril, 2015 07 27;9(2):221-9.
PMID: 26246881

BACKGROUND: Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos.
MATERIALS AND METHODS: In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test.
RESULTS: A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001).
CONCLUSION: Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification.
Fulltext Reliability and construct validity of the Malay version of Theory of Planned Behaviour (TPB) for smoking cessation

Ruslan NH, Yasin SM, Isa MR, Mohd Nasir N, Ahing T, Rajikin MH

Asian Pac J Cancer Prev, 2018 Oct 26;19(10):2815-2820.
PMID: 30361039 DOI: 10.22034/APJCP.2018.19.10.2815

Objective: The aim of this study was to evaluate the psychometric properties of the Malay translated version of the Theory of Planned Behavior (TPB) intention to quit smoking questionnaire.
Methods: A cross-sectional study was performed involving 185 male smokers. The forward-backward translation procedure was adopted to translate the questionnaire from English to Malay. The internal consistency and stability were assessed using Cronbach’s alpha and a correlation analysis and Exploratory Factor Analysis was conducted.
Result: The translated questionnaire showed good internal consistency with Cronbach’s alpha values of 0.86, 0.64, 0.74 and 0.90 for each of the four respective factors. The test-retest reliability revealed acceptable stability, with Spearman’s correlation coefficients ranging from low to moderate (r>0.30-0.50) and a satisfactory inter class correlation coefficient (ICCs). The construct validity achieved an acceptable factor loading for each construct which ranged from 0.40 to 0.90.
Conclusion: The current study provided
psychometric evidence for an appropriate, reliable and valid tool of TPB Malay version. This questionnaire could be applied in evaluating smoking cessation interventions in Malaysia.
Effects of Nicotine and Tocotrienol-Rich Fraction Supplementation on Cytoskeletal Structures of Murine Pre-Implantation Embryos

Hamirah NK, Kamsani YS, Mohamed Nor Khan NA, Ab Rahim S, Rajikin MH

Med Sci Monit Basic Res, 2017 Dec 08;23:373-379.
PMID: 29217815

BACKGROUND Cytoskeletal structures, in particular actin and tubulin, provide a fundamental framework in all cells, including embryos. The objective of this study was to evaluate the effects of nicotine, which is a source of oxidative stress, and subsequent supplementation with Tocotrienol-rich fraction (TRF) on actin and tubulin of 2- and 8-cell murine embryos. MATERIAL AND METHODS Thirty female Balb/C mice were divided into 4 groups: Group 1 received: subcutaneous (sc) injection of 0.9% NaCl; Group 2 received sc injection of 3.0 nicotine mg/kg bw/day; Group 3 received 3.0 sc injection of nicotine mg/kg bw/day +60 mg/kg bw/day TRF; and Group 4 received 60 sc injection of TRF mg/kg bw/day for 7 consecutive days. The animals were superovulated with 5 IU PMSG followed by 5 IU hCG 48 h later. Animals were cohabited with fertile males overnight and euthanized through cervical dislocation at 24 h post coitum. Embryos at the 2- and 8-cell stages were harvested, fixed, and stained to visualize actin and tubulin distributions by using CLSM. RESULTS Results showed that at 2-cell stage, actin intensities were significantly reduced in the nicotine group compared to that of the control group (p<0.001). In Group 3, the intensity of actin significantly increased compared to that of the nicotine group (p<0.001). At 8-cell stage, actin intensity of the nicotine group was significantly lower than that of the control group (p<0.001). The intensities of actin in Group 3 were increased compared to that of nicotine treatment alone (p<0.001). The same trend was seen in tubulin at 2- and 8-cell stages. Interestingly, both actin and tubulin structures in the TRF-treated groups were enhanced compared to the control. CONCLUSIONS This study suggests that TRF prevents the deleterious effects of nicotine on the cytoskeletal structures of 2- and 8-cell stages of pre-implantation mice embryos in vitro.
Deleterious effects of nicotine on the ultrastructure of oocytes: role of gamma-tocotrienol

Rajikin MH, Latif ES, Mar MR, Mat Top AG, Mokhtar NM

Med Sci Monit, 2009 Dec;15(12):BR378-83.
PMID: 19946227

Previous studies have shown that nicotine enhances oxidative DNA damage and leads to increased lipid peroxidation, which affects embryo development. The present study investigated the effect of daily supplementation of gamma-tocotrienol on oocytes of nicotine-treated mice.
Fulltext Nicotine-induced cessation of embryonic development is reversed by γ-tocotrienol in mice

Kamsani YS, Rajikin MH, Mohamed Nor Khan NA, Abdul Satar N, Chatterjee A

Med Sci Monit Basic Res, 2013 Mar 06;19:87-92.
PMID: 23462735 DOI: 10.12659/MSMBR.883822

BACKGROUND: This study aimed to evaluate the adverse effects of various doses of nicotine and protective effects of different concentrations of gamma-tocotrienol (gamma-TCT) on in vitro embryonic development and lipid peroxidation in mice.
MATERIAL AND METHODS: A) Effects of various doses of nicotine on in vitro embryonic development: Female mice were treated with 1.0, 3.0, or 5.0 mg/kg/day nicotine for 7 consecutive days. Animals were superovulated, cohabited overnight, and sacrificed. Embryos were cultured in vitro. Plasma was assayed. B) Effects of concomitant treatment of nicotine concurrently with various doses of gamma-TCT on in vitro embryonic development: Female mice were treated with nicotine (5.0 mg/kg/day), gavaged gamma-TCT of 30, 60, or 90 mg/kg/day or nicotine concurrently with gamma-TCT of 3 different doses for 7 consecutive days. Animals were superovulated, cohabited overnight, and sacrificed. Embryos were cultured and plasma was assayed.
RESULTS: A) Effects of various doses of nicotine on in vitro embryonic development: Number of hatched blastocysts decreased in 1.0 and 3.0 mg/kg/day nicotine groups. Nicotine at 5.0 mg/kg/day stopped embryo development at morula. MDA concentrations increased following all nicotine doses. B) Effects of concomitant treatment of nicotine concurrently with various doses of gamma-TCT on in vitro embryonic development: Embryo development was completed in all groups. MDA concentration increased only in the group treated with nicotine concurrently with 30 mg/kg/day gamma-TCT.
CONCLUSIONS: Nicotine impairs in vitro embryo development and increases MDA in plasma. The deleterious impact of nicotine on embryo development is reversed by supplementing gamma-TCT concurrently with nicotine.
Fulltext Effect of DNase treatment on RNA extraction from preimplantation murine embryos

Norhazlin J, Nor-Ashikin MN, Hoh BP, Sheikh Abdul Kadir SH, Norita S, Mohd-Fazirul M, et al.

Genet. Mol. Res., 2015;14(3):10172-84.
PMID: 26345954 DOI: 10.4238/2015.August.28.1

The quality of RNA is crucial when performing microarray experiments. This is particularly important when dealing with preimplantation embryos, from which a minimum yield of RNA of good quality can be produced. We report the optimization of several RNA extraction methods applied to preimplantation embryos at different stages of development. The quality of the samples was confirmed using a microarray and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. A total of 30 cultured two-cell stage embryos of ICR mice were pooled at the 8-cell, morula, and blastocyst stages. The embryos were divided into two groups comprising DNase-treated and non-DNase-treated RNA samples. Total RNA was extracted using a Pico Pure RNA Isolation Kit following the manufacturer protocol, with some modifications. Lysed samples were bound to a silica-based filter, treated with deoxyribonuclease I (DNase I), and washed several times before elution. RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer and an RNA 6000 Pico Assay kit. Although concentrations of non-DNase-treated RNAs were higher than DNase-treated RNA, DNase-treated RNA gave a higher RNA integrity number compared with non-DNase-treated RNA. Inclusion of DNase treatment in the RNA extraction procedure gave the best quality RNA samples from preimplantation embryos, as validated by microarray and RT-qPCR quality control.
Fulltext Cytoskeletal alterations in different developmental stages of in vivo cryopreserved preimplantation murine embryos

Dasiman R, Rahman NS, Othman S, Mustafa MF, Yusoff NJ, Jusoff WH, et al.

Med Sci Monit Basic Res, 2013 Oct 04;19:258-66.
PMID: 24092420 DOI: 10.12659/MSMBR.884019

BACKGROUND: This study aimed to investigate the effects of vitrification and slow freezing on actin, tubulin, and nuclei of in vivo preimplantation murine embryos at various developmental stages using a Confocal Laser Scanning Microscope (CLSM).
MATERIAL/METHODS: Fifty female mice, aged 4-6 weeks, were used in this study. Animals were superovulated, cohabitated overnight, and sacrificed. Fallopian tubes were excised and flushed. Embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing. Fixed embryos were stained with fluorescence-labelled antibodies against actin and tubulin, as well as DAPI for staining the nucleus. Labelled embryos were scanned using CLSM and images were analyzed with Q-Win software V3.
RESULTS: The fluorescence intensity of both vitrified and slow-frozen embryos was significantly lower for tubulin, actin, and nucleus as compared to non-cryopreserved embryos (p<0.001). Intensities of tubulin, actin, and nucleus in each stage were also decreased in vitrified and slow-frozen groups as compared to non-cryopreserved embryos.
CONCLUSIONS: Cryopreservation of mouse embryos by slow freezing had a more detrimental effect on the actin, tubulin, and nucleus structure of the embryos compared to vitrification. Vitrification is therefore superior to slow freezing in terms of embryonic cryotolerance.

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