P~rtani.ka 8(3),417 -424 (1985)

Histological Changes in the Pregnant, Lactating and InvolutingMammary Gland of the Guinea Pig

W. NORDIN and C.S. LEE 1

Department of Animal Sciences,Faculty of Veterinary Medicine and Animal Sciences,

Universiti Pertanian Malaysia,43400 Serdang, Selangor, Malaysia.

Key words: Histology; mammary gland; leucocytes; guinea pigs.

ABSTRAK

Perubahan histologi kelenjar susu t'ikus belanda pada pertengahan dan akhir kebuntingan, 1

hari selepas beranak, kemuncak laktasi dan 2, 3, 4, 8 dan 12 hari selepas cerai susu telah dikaji. Tum­besar tisu kelenjar berlaku dengan cepat pada akhir kebuntingan hingga ke kemuncak laktasi. Sel-selplasma didapati banyak di ruang antara alveolus dari pertengahan kebuntingan hingga laktasi.Involusi kelenjar susu ialah proses otoZitik yang dibantu oleh sel-sel makrofaj, diiringi denganalveolus-alveolus yang kecut dan bertambahnya tisu perantaraan antara alveolus. Ciri inijelas padahari ke 3 selepas cerai susu dan ia bermula dari periferi kelenjar susu. Sel-sel makrofage terdapatdengan banyaknya dalam tzsu perantaraan dan dalam saluran alveolus dan duktus alveolus. Padahari ke 8 alveolus-alveolus tidak dapat lagi dikenalz' dan pada hari ke 12 selepas cerai susu hanya szsa­szsa saluran dan alveolus sahaja yang kelihatan.

ABSTRACT

The histological changes of mammary glands ofguinea pigs during mid and late pregnancy, 1day after parturition, peak lactation and 2, 3, 4, 8 and 12 days after weaning were studied. Rapidgrowth of the glandular tzssue occurred towards late pregnancy and was completed by peak lactation.Plasma cells occurred in large numbers in the interalveolar connective tzssue during the period be­tween mid-pregnancy and lactation. Mammary involution was an autolytic process aided by the actionof macrophages accompanied by shrunken alveoli and increased interalveolar connective tzssue. Thzsfeature was evident at 3 days after weaning which began from the perzphery of the glands. Thenumber of macrophages were abundant in the connective tzssue and alveolar and ductal lumina. Byday 8, the alveoli were no longer recognisable and at 12 days after weaning only remnants ofductulesand alveoli were present.

INTRODUCTION

The mammary gland is a modified cuta­neous organ (Sisson 1975) with a wide diversity ofphysiological and biochemical functions. Thehistological structure of this organ variesthroughout the productive life of an animal. Thehistology of the mammary gland during the

productive life of the cow (Feldman, 1961),sheep (Lee and Lascelles, 1970) and rat (Jeffers,1935a; 1935b Tateyama et al., 1981) have beenstudied. However relatively little work has beendone on the histology of guinea pig's mammarygland during its productive life. The aim of thepresent investigation is to give a detaileddescription of the histological changes of

IDepartment of Yeterinary Preclinical Sciences, University of Melbourne, Parkville 3052, Australia.

W. NORDIN AND C.S. LEE

mammary gland of the guinea pig during welldefined stages or pregnancy, lactation andinvolution with emphasis mad<; on cell popula­tion at each stage.

MATERIALS AND METHODS

Anz'mals

A total of 18 guinea pigs of Dunkin-Hartleystrain were used in these experiments, 6 of whichwere pregnant and the remaining 12 were lac­tating. Two each of the pregnant guinea pigswere sacrificed at 30 - 32 days of pregnancy (mid­pregnancy) two at 55 - 60 days of pregnancy(late pregnancy) and at 1 day after parturition.

Of the 12 lactating guinea pigs, 2 werekilled at the peak of lactation that is 6 --8 daysafter parturitien. The remaining 10 guinea pigswere separated from their pups at the peak oflactation to initiate involution of the mammaryglands and two in each group were killed at day2, 3, 4, 8 and day-12 after weaning.

All the guinea pigs were killed by intra­peritoneal administration of an overdose of 2~%ethyl carbonate (BDH, United Kingdom). Themammary glands were fixed by perfusionthrough the external pudendal arteries with 4%glutaraldehyde.

SampHng of Tissue

Each mammary gland was divided into 2equal halves. One half was used for frozen andaraldite sections. The other half was furtherdivide-d into ~ equal blocks, from mediallaterally, and used for paraffin sections.

Araldite Sectz'ons

Tissues were post-fixed in 4% glutaral­dehyde in 0.1 M phosphate buffer at pH 7.2 with2% sucrose. The tissues were further processedat room temperature. through 1% osmium te­troxide in 0.1 M-veronal acetate buffer at pH 7.2for I hr. Tissues were then stained en bloc ip. 2%uranyl acetate in water for 30 min., dehydratedin ethanol and embedded in Araldite. Semi-thin

sections were stained with Jeon's stain aeon,1965) for light microsocopy.

Paraffin and Frozen Sectt.'ons

Paraffin sections 7 urn thick were stainedwith Erlich's haemataxylin and eosin, andFeulgen stain (Pearse, 1968). Plasma cells andmast cells were differentiated by staining sectionswith Aldan blue and methyl green-pyronin Y(Boxer & Chadwin, 1966). Frozen sections of10 - 15 urn thick were stained with Oil red 0(Culling, 1957).

RESULTS

During mid-pregnancy the mammaryglands were still small. Histologically, lobulescloser to the papillary duct (a single large ductwhich received smaller ducts draining thelobules) were more developed than lobules closerto the periphery of the glands. These lobulesaround the papillary duct consisted of alveoliwhich were slightly distended and their luminafilled with homogenous secretions (Fig. 1).

Fig. 1. Mammary gland of guinea pig in mid­pregnancy.: Note lobules at the.vicz'nity ofthe papillary duct (d). Each lobule isseparated from one another by looseconnectz've tissue. Note the presence ofhomogenous secretions wz'thin the alveolarlumina. Hand £, x 77.

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Alveolar epithelial cells were cuboidal with largespherical nuclei and scanty pyroninophiliccytoplasm. At this time pyroninophilic cells withclock-face nuclei (identified as plasma cells) wereplentiful in the interalveolar connective tissue(Fig. 2).

Fig. 2. ,A lobule wz"th ducts and rudimentary alveoliin mid-pregnant mammary gland of guineapig. Note the clusters of plasma cells(arrows) in the inter alveolar connectivetissue. Araldite section, Azure II, x 194.

Closer to the periphery of the glands, the10buIes were smaller and were separated by widebands of connective tissue. These lobulesconsisted mainly of ductules terminating into afew alveoli (Fig. 3).

By late l'regnancy, most of the lobules inthe mammary glands were well developed. Fatdroplets were observed in the lumina of alveoliand the ducts. Predominantly, neutrophilstogether with foamy macrophages (Nordin andLee, 1982) were found in the lumina. In Oil redo stained sections, the alveolar epithelial cells

Fig; 3. Lobules at the perzphery of the gland aremade up of rudimentary alveoli and ductulesin the mid-pregnant guinea-pig mammarygland. Hand E, x 188.

were seen to contain fat droplets in the apicalregion. Visible cytoplasm (not occupied by fatdroplets) was very pyroniniphilic. The moststriking feature of the epithelial cells was thepresence of numerous mitotic figures at differentstages of division (Fig. 4). Some epithelial cellscontained 2 nuclei, often referred to as binu­cleated cells. The location and incidence ofplasma cells were similar to those observedduring mid-pregnancy.

Twenty four hours after parturItion themammary glands produced copious quantities ofmilk and became grossly distended. Histologi­cally, the alveoli were markedly distended andfilled with secretions (Fig. 5) and a~veolar

epithelial cells appeared stretched and flattened(Fig. 6). Plasma cells were still predominant andoccurred either singly or in clusters of 2 to 3 cellsin close proximity to the basal region of theepithelial cells (Fig. 6). Isolated mitotic figureswere commonly found.

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Fig. 4. Mammary gland ofguinea pig in latepregnancy. Note alveoli with secretionscontaining dark staining material. Note thepresence of numerous mitotic figures (largearrows) in the epithelt'al cells. Some of themcontain fat droplets (small arrows). Aralditesection, Azure II, x 471.

At peak lactation, the mammary glandularstruCture was basically similar to that observed at24 hours after parturition. Two days after wean­ing, the alveoli were still well defined and theirlumina were filled with secretion of frothy ap­pearance. The incidence of neutrophils wassomewhat increased and occasional lymphocytesand foamy macrophages were also observed.Epithelial cells were cuboidal and filled with fatdroplets and eosinophilic granules. Plasma cellwas the main cell type seen in the connectivetissues between the alveoli.

The size of the glands had involuted con­siderably at 3 days after weaning. At the peri­phery of the glands, the alveoli were shrunkenand their lumina filled with distinct fatty secre­tions. Degenerating epithelial cells were a predo­minant feature of the histological picture and intheir lumina, foamy macrophages were more

Fig. 5. Lobules separated by a thin band ofconnective tissue. Note that the markedlydistended alveoli containing milk. 24 hrsafter parturition, Hand E, x 47.

Fig. 6. The alveolar epithelium is flattened. Notethat some plasma cells are located at closeproximity to the basal region of theep£thelium (arrow). 24 hrs after parturz'tion,Hand E, x 471.

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Fig. 7. Alveoli (a) and a ductule (d) at theperzphery of the gland. Note that thecellular outline of some epithelial cells arenot discernible (large arrows). Within thelumen of the ductule are foamymacrophages and cellular debris (smallarrows). 3 days after weaning, Hand E x471.

commonly seen (Fig. 7). Sections stained withFeulgen stain (Pearse, 1968) revealed that thecells in the lumina were karyorrhectic andpyknotic cells.

Closer to the papillary duct, the alveoli werestill well defined but in most cases they were notfilled with secretion; this apparently had beenremoved during tissue processing (Fig. 8). Largefat droplets were sometimes seen at the apicalregion of the epithelial cells and their nucleiwere larger and stained lightly. In the lumina,foamy macrophages were abundant (Fig. 8).Desquamated epithelial cells and neutrophilswere occasionally observed (Fig. 8).

At 4 days after weaning most of the secretionsin the mammary glands had been resorbed asjudged by the size of the glands. Alveoli werenow difficult to recognise as distinct structures.Epithelial cells contained single fat-filledvacuoles and in the majority of the cells thenuclei were no longer visible (Pig. 9). Connective

Fig. 8. Alveoli located adjacent to the papillaryduct. Note that they are still well defined.Their lumina contain many foamymacrophages (small arrow) and 2desquamated epithelz'al cell (large arrow). 3days after weaning, Hand E, x 471.

Fig. 9. A lveoli with epithelial cells containing largefat droplets. In the lumina are presentnumerous macrophages (small arrows) and2 - 3 desquamated epithelial cells. (largearrow) 4 days after weaning, Aralditesection, Azure II, X 486.

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tissues betw~en the alveoli and lobules ,were nowmore distinct. Cells with oval and irregularshape with alcianophilic cytoplasm "(referred asmast cells) were commonly found in the connec­tive tissue in close association with the plasmacells. Foamy macrophage was the main cell typeseen in the lumina of alveoli and ducts (Fig. 9).

"This cell was also seen in the lymphatics (Fig.10).

Eight days after weaning the glands werealmost involuted. Alveolar remnants were seento contain degenerating cells and large cells witheosinophilic cytoplasm. These cells were alsofound in the connective tissue {Fig. 11). By now,plasma cells were very few in number.

Twelve days after weaning, the degenerat­ing cells had virtually disappeared and thealveolar and ductular remnants were lined with1 or 2 layers of closely packed cuboidal epithelialcells (Fig. 12). Foamy macrophages were seldomseen at this time. However, large cells witheosinophilic cytoplasm" were commonly seen.

Ft'g. 10. A large lymphatt'c vessel contat'nt'ngmacrophages and lymphocytes t'n z'tslumen. 4 days after weant'ng. A raldt'tesectt'on, Azure II, X 486.

F£g: 11. A lobule wt'th alveolt' difft'cult to recognz'zeas dt'stz'nct structures. Wt'tht'n the remnantsof the alveolz' and t'n the connectt've tt'ssueare present large eost'nophzHc cells (arrows).8 days after weant'ng, Hand E, x 486.

F£g. 12. Showt'ng markedly shrunken lobulescontat'nt'ng mat'nly ductular and alveolarremnants. 12 days after weant'ng, Hand E,x 192.

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DISCUSSION

At mid-pregnancy the lobular developmentwas only concentrated around the papillaryduct. During this stage the alveolar epithelialcell cytoplasm had already started synthesisingprotein as evident from its pyronin stain affinity.Secretory activity of these alveoli had also startedat this time as their lumina were filled withhomogenous secretions.

As parturition approached there was arapid development of the alveoli as apparentfrom the numerous mitotic figures present in thealveolar epithelium. Most of the lobules werewell developed at this stage and secretory activityof these alveoli had been very vigorous. Thealveolar epithelial cell cytoplasm had started tosynthesise fat and the alveoli showed a strikingenlargement due to the accumulation of secre­tions containing numerous fat droplets. This issimilar to the mouse, where the synthesis of fatalso occurred at the end of pregnancy (Tate­yamaetal.,1981).

After parturition very few isolated mitoticfigures were present in the alveolar epitheliumwhich suggested that alveolar development atthis time was minimal. This pattern of lobulardevelopment after parturition is similar to thatdescribed by Nelson et ai. (1962), in rat(Munford, 1964), in sheep (Anderson, 1979) andin goat (Anderson et ai., 1981).

, The fatty vacuolation and other degenera­tive .changes in the alveolar epithelial cellsobserved as involution progressed was essentiallysimilar to those described by other workers whostudied involution in rats Geffers, 1935a, 1935b),guinea-pigs (Hessel1;>erg and Loeb, 1937) andsheep (Lee et ai., 1969; Lee & Lascelles, 1970).On the second day abundant eosinophilicgranules accumulated within the cytoplasm ofthe alveolar epithelium. ~is was probablyprotein materials which were not secreted by thetells due to impedance of the secretory mecha­nisms of the cells as a result of the increased.intramammary pressure. Between 3 and 8 daysafter weaning, the epithelial cells had lost theirinternal structure; the nucleus became pyknotic

and eventually disappeared while the cytoplasmwas ultimately replaced by fat. By the twelfthday, the glandular tissue had completely involut­ed leaving only alveolar and ductular remnants.

Large number of plasma cells were observedin the inter alveolar connective tissue during midand late pregnancy, lactation and early involu­tion. These cells could be active producers ofIgA as this class of immunoglobulin has beenreported to be the major class in mammarysecretions of this species (McDowell et al., 1971).However, in contrast to the sheep, large numbersof lymphocytes in the interlobular connectivetissue during late pregnancy, lactation and invo­lution were observed (Lee et ai., 1969; Lee andLascelles, 1970).

The foamy macrophages observed withinthe alveoli and ducts during pregnancy couldhave migrated from the surrounding connectivetissue in response to the large amount of fat inthe colostrum. During the course of involutionthese cells persisted in the alveolar and ductallumina. Lee & Lascelles (1970) made a similarobservation in the mammary glands of sheep. Itis interesting to note that these cells were seldomseen in the glands at the advance stage of involu­tion where alveoli were mostly dry. This furthersuggests that the foamy macrophages may havemigrated into the lumina only in response to thefat and cellular debris.

ACKNOWLEDGEMENTS

We would like to thank Miss C. Byrns andMiss A. Veenstra for their technical assistance.This work was support~d by grants from theUniversity of Melbourne.

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(Recez'ved 23 May, 1985)

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