The Effect of L-Arginine and Antagonist L-Name in the Mammary Gland Of

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

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:

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 dephosphorylation. Upregulationsetpoint of L-arginine-nitric oxide induced estrogen may be needed for the synthesis of growth factors and progesterone receptor(Franklyn and Bolander, 2000).

One of the excellent studies indicated the positive effect of several hormones mediationfor the development of the mammary glands fractions was done by Franklyn and Bolander, (2000). These were usher mainly prolactin synthesis and secretion. The increasing blood concentrations of estrogen during late pregnancy appeared to be responsible for the elevated levels of prolactin narrow limited value, which are necessary to prepare the mammary gland for lactation at the end of gestation (Franklyn and Bolander, 2000).

Furthermore, the upsurge of estrogen in L-arginine treatment triggered of insulin-like growth factor-I (IGF-I) cascade on mammary development and growth hormone-induced IGF-I messenger ribonucleic acid that was used a marker of proliferation events (Ranga, 2010).

JPCS Vol(6) ● Jan-March 2013 Alkareemet al. ● The Mammary Gland of Pregnant Mice

2ndProgesterone, the progesterone hormone was assigned the exaggerated role in developing and enlarged structural components that may be biased results of the attribution to direct effect of L-arginine as to modify mammary gland structuresin addition to the dominant effect on ductal system of the mammary gland stated in previous studies (Russo and Russo, 1993;Feng et al., 2007 and(Mueller et al,. 2002). Also, an increase in progesterone under loading dose of L-arginineresults in the dominancy of the progesterone which rebooted the mammary gland growth during pregnancy via induce differentiation of terminal end buds and terminal ducts (Russo and Russo, 1993 and Fenget al., 2007),in addition to the lobulo-alveolar differentiation in the mammary gland at pregnant term.

3rdProlactin,the estrogen mediated prolactin release acts as a signaling for the proliferation and functional differentiation of lobulo-alveolar structures during late pregnancy and lactation periods (Topper and Freeman, 1980). Several notions in the available literatures referred to direct stimulation of L-arginine-NO pathway to prolactin synthesis and release (Bolander, 2002).This clear evidence was resulted to the attribution of NO to the superior lobulo-alveolar growth of the mammary gland. The narrow limit increase of prolactin due to loading dose of L-arginine enhanced molecular cellular event to result in increased growth mammary gland.Prolactin plays a crucial role in the early stage of alveolar proliferation via increase donation of NO with the latter presumably act on two separated pathways to control the mammary epithelium hypertrophy and increase densities through: 1. stimulates mammary gland tissues growth via Jun N-terminal kinase(JNK) of the protein kinase G as a cellular second messenger which were a key-lock of cell cycle G0 and G1 initiator mediated through DNA-RNA (Bolander, 2002)and 2. The prolactin up-regulated levels under L-arginine donated nitric oxide self-control of increase cellular growth inhibits cellular differentiation throughExtracellular Signal-Regulated kinase (ERK) (Bolander, 2002) over growth, which suggestschanges of the Jun N-terminal kinase (JNK) then blocked the effect of NO on DNA synthesis. Furthermore, other suggestion may be explained withthe increase of ductal alveoli densities and numbers(Mammarytree growth, length of the mammary gland, glandular volume density) in late pregnancy and lactation period under L-arginine loading dose, the prolactin could also enhance DNA synthesis stimulated by sub maximal prolactin concentrations(Franklyn and Bolander, 2000). The prolactin presumably reload the effect L-arginine-NO through elevation of calcium ions level (Bolander, 1985) which were mediated the increase in both arginine transport(Rillemaand Koduri1992) and PKB activity (Al- Sakkafet al.,2000 and Tessieret al.,2001). The pathway of L-arginine-induced prolactin may be attributed to the provoked activation of STAT5 in the mammary gland epithelium which is associated with the growth hormone(Rillema and Koduri,1992).STAT5a is essential for prolactin-induced terminal differentiation of the mammary gland during pregnancy and lactation (Kazanskyet al., 1995 and Li et al., 1997).

Figure 3:A scheme illustrating the possible mechanisms by which PRL could activate NO synthase and the location where various compounds used in thisstudy act. DAG, diacylglycerol; PDK1, phosphatidylinositol-dependent proteinkinase 1; PI(3)Pn, phosphatidylinositol 3′-phosphate; PLCγ, phospholipaseCγ; PP2B, protein phosphatase 2B; TPA, a phorbol ester(Bolander, 2002).

4th Growth hormones; a number of projects attributed the effect of L-arginine on the mammary gland structuresof a regulating role and maximized capacitation growth of cellular then mammary gland components.Therefore, L- arginine loading dose stimulated the secretion of growth hormone works subsidiary via boosting of local production of insulin-like growth factor I (IGF-I) within the mammary gland which play a vital modification of DNA-RNA or essential for rodent mammary gland development through stimulation of mRNA expression. On the other hand, one

JPCS Vol(6) ● Jan-March 2013 Alkareemet al. ● The Mammary Gland of Pregnant Mice

of the important effects of GH is soundness ductal elongation and reliable duplication of layers differentiation of ductal epithelia into terminal end buds (Coleman et al., 1988).

5thOther factor; the cellular mechanism by which mammary growth occur during lactation in response to increased suckling intensity are controlled by both the autocrine and endocrine functions. The fetuses under loading dose of L- arginine have increase in their weight and that may also correlate with suckling mechanismthus led to enhance mammary gland functional parts during lactation period (Wilde et al., 1989). L-arginine –nitric oxide is particularly involved not only in lactation but also in preparatory changes of the mammary gland via increase blood follow and blood volume which were conditioner player to mammary gland growth, then, mammary gland feeding, as well as erection of the nipple and the areola, as well as enlargement of the mammary ducts, which usually occur during the early postpartum phase (Franklyn and Bolander, 2000). An important issue which remains to be addressed is whether L-arginine donated NO simply accelerated the pace of mammary gland development (Figures 7, 8, 9 and 10) or whether L-arginine during pregnancy is able to imprint a durable activator effect on maturation and proliferative potential of the mammary epithelium. In other words, will the mammary gland in L-arginine dosing eventually catch up and differentiate fully to a stage comparable with that seen in control mice?. It presumably would be reasonable to expect that the biosynthesis of eicosanoids will be affected by the up-regulated availability of arachidonic acid in the mammary gland tissues under provocation target phospholipids of membranous bilayers (Robinson, 1999). In addition to mammary gland tissue development motivation, L-arginine is known to modulate immune functions, atherogenesis and phorbol ester-mediated events in keratinocytes.These complementary factors resulted in progressive expansion of the mammary gland could be involved in a causal sequences of L-arginine dosing. It is well established that epithelial/mesenchymal interactions are important for true actual development of the mammary ductal tree and its differentiation during pregnancy into a milk producing structure in lactation term (Robinson, 1999). The L-arginine increases density of mesenchyme are presented inTables 1 and 3.Thismesenchymal tissue contains a heterogeneous group of cells which includes several fat cells and fibroblasts which are capable of producing factors that can promote the growth of the epithelial cells which may be reflected in their increment of TEBs branching and density (Sternlicht, 2006). Previous studies hypothesized that L-arginine donated NO promoted mechanism included two migrant stromal cells recruited around the growing ducts, macrophage and eosinophil, and showed that these cells played an important role in the development of the mammary gland. Furthermore, several studies revealed the direct effect of L-arginine nitric oxide on the macrophages and eosinophils, whose recruitment to the mammary gland during pregnancy and lastly in lactation period closely paralleled to the formation and outgrowth of TEBs. These cells were found in other regions but also in overlapping positions around the TEBs, with eosinophil's mostly around the cap cells, and macrophages along the shaft immediately adjacent to the TEB. TEB formation is essential for ductal elongation in mammary gland development, since the undifferentiated cells located in the outer layer of the TEB structure, known as ‘cap cells’ may be able to give rise to the intermediate, luminal and myoepithelial cells of the advancing ducts (Williams and Daniel, 1983).

Table 1 Effect of L-arginine1 and L-NAME2 on the percentage of glandular volume density in mammary gland of the pregnant and lactating mice

Glandular volume density %

Pregnancy Lactation Control Normal saline 11.88 ± 2.51ABa 16.40 ± 0.19Ab 100 12.15 ± 1.27Ba 23.92 ± 0.98Bb L-arginine 150 23.11 ± 0.56Ca 25.81 ± 1.43Bb 200 47.80 ± 0.78Da 82.70 ± 3.11Cb 250 83.69 ± 0.40Ea 75.32 ± 7.04Db 300* 10.26 ± 1.13Aa 11.55 ± 1.31Fa L-NAME 100 4.21 ± 0.29Fa 7.05 ± 1.15Gb Number of mice per dosed group =10 pregnant. Different capital letters denoted significant difference (p< 0.05) between dosed groups. Different small letters denoted significant difference (p< 0.05) between lactation and pregnancy period. 1 L- arginine dosed: 100, 150, 200, 250 and 300 mg/kg BW, orally, daily from 0-18 days of pregnancy period and from 1-21days of lactation period. 2 L-NAME groups dose100 mg/kg B.W IP, daily from 0-18 days of pregnancy period and from 1-21days of lactation period. *aborted mice.

JPCS Vol(6) ● Jan-March 2013 Alkareemet al. ● The Mammary Gland of Pregnant Mice

Table 2 Effect of L-arginine1 and L-NAME2 on the numbers of terminal end bud/mm2 in the pregnant and lactation mice.

Numbers of the terminal end bud

Pregnancy Lactation Control Normal saline 7.03 ± 1.06Aa 9.28 ± 0.23Ab 100 14.28 ± 1.53Ba 18.04 ± 1.22BCb 150 13.11 ± 0.28Ba 19.96 ± 2.09Bb L-arginine 200 16.80 ± 0.76Ca 20.42 ± 3.12CDb 250 10.85 ± 0.41Da 21.59 ± 0.83Db 300* 6.32 ± 1.28Fa 1.71 ± 1.61Eb L-NAME 100 0.79±0.033Ga 0.20 ± 0.003Fb Number of mice per dosed group =10 pregnant. Different capital letters denoted significant difference (p< 0.05) between dosed groups. Different small letters denoted significant difference (p< 0.05) between lactation and pregnancy period. 1 L- arginine dosed: 100, 150, 200, 250 and 300 mg/kg BW, orally, daily from 0-18 days of pregnancy period and from 1-21days of lactation period. 2 L-NAME groups dose100 mg/kg B.W IP, daily from 0-18 days of pregnancy period and from 1-21days of lactation period. *aborted mice.

Table 3 Effect of L-arginine1 and L-NAME2 on the Percentage of alveolar buds density in mammary gland of the pregnant and lactation mice

Alveolar buds density %

Pregnancy Lactation Control Normal saline 0.20 ± 0.007Aa 0.28 ± 0.006AFb 100 0.32 ± 0.036Ba 0.27 ± 0.015AFb 150 0.44 ± 0.024Ca 0.49 ± 0.036Ba L-arginine 200 0.79 ± 0.030Da 0.85 ± 0.102Cb 250 0.35 ± 0.012Ba 0.40 ± 0.017Da 300* 0.32 ± 0.005Ba 0.22 ± 0.008Fb L-NAME 100 0.12 ± 0.0020Ea 0.11±0.0023Aa Number of mice per dosed group =10 pregnant. Different capital letters denoted significant difference (p< 0.05) between dosed groups. Different small letters denoted significant difference (p< 0.05) between lactation and pregnancy period. 1 L- arginine dosed: 100, 150, 200, 250 and 300 mg/kg BW orally, daily from 0-18 days of pregnancy period and from 1-21days of lactation period 2 L-NAME groups dose100 mg/kg B.W IP, daily from 0-18 days of pregnancy period and from 1-21days of lactation period. *aborted mice

Table 4. Effect of L-arginine1 and L-NAME2 on the Percentage of mammary growth tree in mammary gland of the pregnant and lactation mice

Mammary growth tree %

Pregnancy Lactation Control Normal saline 21.43 ± 2.45Aa 34.78 ± 4.44Ab 100 43.76 ± 6.01Ba 51.13 ± 2.50Bb 150 56.31 ± 4.96Ca 68.78 ± 4.30Cb L-arginine 200 89.24 ± 7.23Da 95.06 ± 7.65Db 250 77.88 ± 5.83Ea 90.62 ± 8.30Fb

300* 19.56 ± 1.18Aa 10.89 ± 1.19Gb L-NAME 100 9.42 ± 1.05 Aa 2.86 ± 0.49 Ha Number of mice per dosed group =10 pregnant. Different capital leitters denoted significant difference (p< 0.05) between dosed groups. Different small letters denoted significant difference (p< 0.05) between lactation and pregnancy period. 1 L- arginine dosed: 100, 150, 200, 250 and 300 mg/kg BW, orally, daily from 0-18 days of pregnancy period and from 1-21days of lactation period 2 L-NAME groups dose100 mg/kg B.W IP, daily from 0-18 days of pregnancy period and from 1-21days of lactation period *aborted mice

JPCS Vol(6) ● Jan-March 2013 Alkareemet al. ● The Mammary Gland of Pregnant Mice

Table 5.Effect of L-arginine1 and L-NAME2 on the Length of mammary gland ducts tree in mammary gland of the pregnant and lactating mice

Length of mammary gland ducts mm

Pregnancy Lactation Control Normal saline 16.32± 1.75Aa 19.69 ± 1.80Ab 100 17.05 ± 2.81Aa 24.59 ± 3.45Bb 150 21.91 ± 1.52Ba 27.20 ± 1.36Cb L-arginine 200 36.72 ± 3.40Ca 39.51 ± 1.14Db 250 39.84 ± 1.41Da 35.62 ± 3.33Fb 300* 12.25 ± 0.86Fa 4.28 ± 0.03Gb L-NAME 100 5.99 ± 0.31 Ga 1.00 ± 0.04Hb Number of mice per dosed group =10 pregnant. Different capital letters denoted significant difference (p< 0.05) between dosed groups. Different small letters denoted significant difference (p< 0.05) between lactation and pregnancy period. 1 L- arginine dosed: 100, 150, 200, 250 and 300 mg/kg BW. Orally, daily from 0-18 days of pregnancy period and from 1-21days of lactation period 2 L-NAME groups dose100 mg/kg B.W IP, daily from 0-18 days of pregnancy period and from 1-21days of lactation period. *aborted mice

Figure 4.Mammary gland system in control group during pregnancy period Tertiary terminal ducts (18 days) showing normal number of the ducts and terminal end buds. Carmine red, Mag. 400X. Secondary terminal ducts

Terminalend bud

1 Primary terminal ducts

Figure 5.Mammary gland system in the control group during lactation period (21day) showing normal number of ducts (primary, secondary and tertiary) and terminal end buds. Carmine red, Mag. 100 X.

JPCS Vol(6) ● Jan-March 2013 Alkareemet al. ● The Mammary Gland of Pregnant Mice

Figure 6.Mammary gland system in L- arginine dose 200mg/kg BW during pregnancy period (18 day) showing increased terminal end buds. Carmine red, Mag. 100X.

Figure 7.Mammary gland system in L- arginine dose 200 mg/ kg BW during lactation period (21day) showing increased primary, secondary and tertiary terminal ducts and decreased terminal end buds. Carmine red, Mag. 100X.

JPCS Vol(6) ● Jan-March 2013 Alkareemet al. ● The Mammary Gland of Pregnant Mice

Figure 8.Mammary gland of control pregnant mouse. a. Skin (stratified squamous epithelium) b. superficial dermal layer (relatively thick connective tissue) c. external oblique abdominal muscle d. superficial layer of connective tissue mammary lamina e. mammary alveoli (glandular tissue) f. adipose tissue g. glandular duct h. blood vessel i. deep layer of mammary lamina j. abdominal musculature k. mammary lymph node (supra-mammary) l. papillary duct. lactiferous sinus. H. & E. stain, Mag. 140.

Figure 9. Mammary gland of control lactating mouse. Normal alveolar globular appearance and mild ductal milk fullness as well as apoptotic cell in different parts. H. &E. Stain. Mag. 200X

JPCS Vol(6) ● Jan-March 2013 Alkareemet al. ● The Mammary Gland of Pregnant Mice

Figure 10.Mammary gland of pregnant mouse treated with 200 mg/kg body weight of L arginine during gestation period. Notice the amount of brown (arrow) and white adipose tissue (f) relative to the glandular tissue (e) at this stage and the ducts filled by milk secretion and clearly the found fat globular droplets in milk secretion containing ducts. H. &E.stain;Mag140X.. 140 X.

Figure 11. Mammary gland of lactating mouse given 200 mg/kg body weight L arginine during gestation period. Notice well developed glandular tissue as well as ductal system of the gland with secretory materials inside the lumen. Notice that very little adipose tissue is present within the gland at this stage. H.& E. Stain; Mag. 200X.

JPCS Vol(6) ● Jan-March 2013 Alkareemet al. ● The Mammary Gland of Pregnant Mice

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