JPCS Vol(6) ● Jan-March 2013 www.arpapress.com/Volumes/JPCS/Vol6/JPCS_6_04.pdf THE EFFECT OF L-ARGININE AND ANTAGONIST L-NAME ON THE MAMMARY GLAND OF PREGNANT MICE Marwa Abdul Alkareem, Mohanad A. AlBayati1& WaelKhamas2 1University of Baghdad: College of Veterinary Medicine Department of Physiology and Pharmacology, Iraq 2Western University of Health Sciences: College of Veterinary Medicine, CA. Pomona, USA. ABSTRACT L-arginine is well known as a precursor of Nitric Oxide (NO). NO is one of a major endothelial-derived relaxing factor listed as an endogenous messenger molecule involved in a variety of dependent physiological events through increasing blood flow then blood volume in tissues. L-arginine promotes various fertility parameters and improves fetal traits by acceleration of dramatic molecular events. All of that resultsin a speculation to have superior pups weight through exaggerated mammary gland function and improve milk quality. This study was performed to pharmacologically enhance the performance of the mammary gland by using L-arginine as a forerunner of NO. The study protocol consisted of a total number of 130 pregnant and lactating mice separated into two main groups equally; each one was randomly divided into 3 subgroups [control, L-arginine and L-NAME (served as NO inhibitor)]. L-arginine dosed orally with the following groups: 100, 150, 200, 250 and 300 mg/kg BW daily in pregnant and lactating mice, L-NAME dose 100 mg/kg BW daily dose IP and normal saline was given to 20 female mice which served as control (10 pregnantand 10 lactating respectively).Four mammary glands were evaluated and then yielded the following:Histological and stereological profiles, Development and branching of the mammary alveoli and the reduction of adipose tissue with profuse milk accumulation were observed. Also, an increase in stereological and morphometrical profiles in L-arginine dosed groups in both pregnant and lactating groups as compared with L-NAME, L-arginine dose 300 and control groups. INTRODUCTION L-arginine is an amino acid, one of the 20 amino acids that serve as the building blocks in proteinsynthesis(Tapiero, 2002). L-arginine derived NO as a polyvalent molecule displayed pivotal key in general fertility. L-arginine-NO presently performimportant role inimproving male fertility for exampleenhance penile erection (AlShaty, 2007and Asker, 2012), enhancement of chemical and physical properties of semen quality (Shaheed, 2010 and AlShaty, 2007). Furthermore, aclear turnover in sexual hormones could regulate ovarian function and superovulation as to extends estrus phase windows for prolonged time of conception in addition to improve fetal traits (Hasan, 2012; Mahdi, 2008; Shakir, 2008 and Asada, 2011).NO acts in uterine quiescence during gestation period (Hasan, 2012 andShakir,2008). Data in scientific literatures implied and pointed to the effect of L-arginine which donated NO which has mammogenic effect and would presumably lead to better quality milk secretion as well as capitalize on fetal traits. Consequently, if L-arginine had a powerful vasodilatation and increase blood volume and blood flow (Hasan, 2012 and Asada, 2011), thenthe speculation of the role to improve the pups weight by improving mammary gland function and milk quantity and quality will be correct. Results from suggested ideas of previous theses and literatures stated that efficient mammary gland performance accompanied withbetter pups' fitness.Results fromthis led to the attempt to exploreand assessthe functional morphometrical and stereological profiles of the mouse mammary gland and their yield and explore the milk secretion quantity and quality in loaded L-arginine doses in mice. MATERIALS AND METHODS Whole mammary gland analysis The partsof the mammary glandswere collected according to Bocchinfuso, et al. (2000) as follow: Procedure steps: Step I:The excised mammary glands of both pregnant and lactating mice were placed on glass slides and immersed in tissue fixative for1 hour at room temperature. Fixative composition concentration% Glacial acetic acid 20 Ethanol 75 26 JPCS Vol(6) ● Jan-March 2013 Alkareemet al. ● The Mammary Gland of Pregnant Mice Step II:The glands were placed in 70% ethanol for 15 min and then rinsed in distilled water for 5 min. Step III:The mammary glands were stained overnight at room temperature in carmine alum solution. Preparation of carmine alum solution Carmine natural red1g and2.5 g Aluminum potassium sulfatedi Step IV: The stained glands were dehydrated progressively in ethanol solution 70, 95 and 100% at 5 minutes for each concentration. Step V: The fat pads of mammary tissues were demulsified in xylene for 15 minutes before mounting on slides. The terminal end bud and ductal area parameters were determined and calculated using40 X lens as impact enlargement factor. Mammary whole mount were examined under light microscope for epithelial growth, length of mammary tree, number of terminal end buds (TEB) and density of alveolar bud. Mammary growth was evaluated as percentage of the mammary epithelium that occupied the mammary fat pad.Mammary gland size was determined by mammary tree length, which was measured as the length in millimeters between the nipple and the most distal terminal branches.The identification of terminal structures was based on the classification by Russo and Russo (1978). Club-shaped terminal ductal structure greater than 100µm in diameter were classified as TEBs, while terminal structures less than 100µm in diameter were considered terminal ducts. ABs were identified as terminal or lateral buds that had differentiated by septation, cleavage and further sprouting into 3- 5 Smaller buds. Scale of ABS AB was assessed qualitatively for density on a scale of 0-3 according to Hilakivi-Clarke et al. (1997). 0=absent1=low2=moderate3=abundant Volume density of acino-tubular structures= points counted over acino-tubular structures/ points counted over acini, duct, adipose tissue, and fibrous stromaX100 (Baak and Oort, 1983). RESULTS AND DISCUSSION The study results showed the end points of L-arginine effect on mammary gland development during gestation and lactation periods as well as its direct contribution to fetal /pups and maternal growth and function. Generally, L-arginine nitric oxide donated is a signal player in cellular functions and systematic modulation according to this fact the prime result displayed the following data: Histological and stereological profile Results exhibited the following stereometrical calculated values of glandular volume density, alveolar bud density, mammary growth tree, number of the terminal end bud and length of the mammary duct respectively, during both pregnancy and lactation periods, which were increased significantly (p<0.05) in L-arginine loading dose at 100, 150, 200, 250 mg/kg BW of pregnancy period (0-18days) and lactation period 1-21days dose as compared with control group(Tables 1,2,3,4, and 5).While L-NAME showed stereometrical parameters which decreased significantly (p<0.05) when compared with both L-arginine dosed groups and control group. Whereas, the dosed group 300 mg/kg B.W orally daily (0-18 days) aborted at 14th to 16th day of pregnancy with pronounced and significant (p<0.05) decrease in all parameters as compared with other L-arginine dosed groups but not the control. These results may be attributed to direct involvement in the molecular events of the mammary gland cells as several notions dealtwith L-arginine promotion of transcription and translation of DNA-RNA and increased functional properties in cells activity. That alone offered an impression like the L-arginine increased DNA-RNA concentrations which they were positively correlated with cellular proliferation and promotion ofthe mammary gland alveolar- ductal enlargement and elongation (Iizuka, 1997). On the other hand, the L-arginine-nitric oxide holds several activities that promoted and provoked the developed stereological profile indirectly through maximized extrinsic and intrinsic cellular events, which were presumably deposit signal pathway modulation of DNA-RNA facilitation and thus up-regulated cell numbers and enlarged histological structures (Franklyn and Bolander, 2000). In summary, several physiological events controlled by L-arginine-nitric oxide include: 27 JPCS Vol(6) ● Jan-March 2013 Alkareemet al. ● The Mammary Gland of Pregnant Mice 1st estrogen, several notions in one literature detected that L-arginine is responsible for a closed regulation effects of the estrogens (Chew, 1983) and enhanced the circulating estrogen levels. However, other study revealed physiologically that estrogen promotes the growth of the alveolar structures during pregnancy through induction of differentiation of terminal end buds (Figure 1). Figure 1.Positive co relation presented by the regression factor (R2) between the number of the terminal end buds and estrogen level under L-arginine dosed in lactating mice. This may be the primary factorwhich suggestedthat L-arginine controls the molecular events of proliferations that depicted in the hypothetical diagram displayed in Figure 2. L-arginine-NO Figure 2: Proposed model for progesterone- dependent endometrial cell proliferation cycle viewing the potential control points for cell cycle progression. Cells are driven into S and M phases by the formation of cyclin–cdk complexes. Progesterone may motivate the synthesis of cyclins in G1 and G2 phases of the cell cycle. Activation of the complex requires kinase binding to cyclin, phosphorylation, and