J. Cell Sci. 72, 185-193 (1984) 185Printed in Great Britain © The Company of Biologists 1984

CENTRIOLES IN THE MAMMARY EPITHELIUM OF

THE RAT

DANIEL P. DYLEWSKI AND THOMAS W. KEENANDepartment of Biochemistry and Nutrition, Virginia Polytechnic Institute and StateUniversity, Blacksburg, VA 24061, U.SA.

SUMMARY

Through serial thin-section analysis of rat mammary epithelial cells, the number of centrioles percell and their intracellular location were determined. In all developmental stages (e.g. virginal,pregnant, lactation, involution), each epithelial cell contained a single centriole that was located inthe apical region. Centrioles were 200-220 (x = 210) nm in transverse section, and exhibited thetypical 9 x 3 'pinwheel' configuration of microtubules. In longitudinal section, centrioles were330—380 (x — 360) nm in length. Each centriole was surrounded by a homogeneous pericentriolarmatrix. During mitosis in pregnant animals, centrioles were paired at the nuclear poles and orientedat right angles (90°) to each other. At the completion of mitosis a single diplosome (pair ofcentrioles) was associated with each interphase nucleus. Because all postmitotic cells contained onlya single centriole, it was assumed that one of the two diplosomal centrioles had disintegrated. Thereappeared to be a correlation between centriole location and cell polarity. When centrioles werelocated near the apical plasma membrane, epithelial cells exhibited polarity. However, whencentrioles were associated with the nuclear poles during mitosis, epithelial cells were typicallyapolar. These observations suggest that centrioles may function as determinants in cell polarity.

INTRODUCTION

Centrioles are ubiquitous organdies found in most living cells with the exceptionof angiosperms, higher gymnosperms, higher fungi and several other groups of organ-isms (Wheatley, 1982). Since their discovery by van Beneden (1876), microscopicinvestigations have led to a thorough understanding of centriole structure. Centriolefunction, on the other hand, has remained poorly understood and is the subject ofcontinuing controversies (for review, see Wheatley, 1982). The intracellular positionof centrioles has led some investigators to believe that they are the primary deter-minants of cell polarity (Mazia, 1978).

Bargmann & Knoop (1959) were the first to describe the fine structure of themammary epithelial cell. Since their work, the morphology, morphometry and intra-cellular distribution of all organelles and cytoplasmic structures have been determinedwith the exception of centrioles (for review, see Hollmann, 1974; Keenan, Morr6 &Huang, 1974). Centrioles of mammary epithelial cells have never been described andtheir intracellular location during cell development is unknown. Ultrastructural in-vestigations of centrioles are needed, however, because they: (1) may lead to a betterunderstanding of structure-function relationships in the mammary epithelial cell;

Key words: centriole, mammary epithelium, cell polarity.

7 CEL72

186 D. P. Dylewski and T. W. Keenan

and (2) may help to resolve the possible role of centrioles as determinants in the

establishment of cell polarity.

The purpose of this study was to describe the ultrastructural morphology of

centrioles and determine their number and intracellular location in rat mammary

epithelial cells during development and differentiation.

MATERIALS AND METHODS

Twenty-one primiparous female Sprague-Dawley rats between 3 and 4 months of age were killedby cervical dislocation. Tissues samples were taken from 12-week-old virgin animals, from animalsat 1, 7, 14 and 21 days pregnant, at day 7 of lactation, and at day 4 of involution. Three animals wereused for each stage. Portions of mammary tissue were dissected from each animal and placedimmediately into large volumes of 0-5 % glutaraldehyde in 0-1 M-cacodylate buffer (pH 7) at roomtemperature and cut into pieces of 1 mm . Specimens were then fixed by the method of Warchol,Herbert & Rennels (1974), which was developed for preservation of microtubules. Tissue sampleswere immersed in 2-5 % glutaraldehyde in 0-1 M-cacodylate buffer (pH 7) at room temperature for1 h, transferred to a cold room at4°C for 1 h, then placed on crushed ice for lOmin. Specimens werepostfixed in solutions (2:1, 1:2, v/v) of 2-5 % glutaraldehyde in 0-1 M-cacodylate buffer, and 1 %osmium tetroxide in 0-1 M-cacodylate buffer (pH 7) for 30min on crushed ice. Finally, tissuesamples were immersed in 1% osmium tetroxide in 0-lM-buffer for 2h on crushed ice, rinsedbriefly in cold buffer, dehydrated in a graded series of ethanol, soaked in acetone, and embeddedin a mixture of Epon-Araldite (Poolswat, 1973). Serial thin sections were collected on Formvar-coated copper slot grids, and stained with uranyl acetate (Watson, 1958) and lead citrate (Venable& Coggelshell, 1965). Sections were examined in a Philips EM300 electron microscope operated at60 kV. All linear dimensions were based on measurements of at least 25 examples.

RESULTS AND DISCUSSION

During the development of the rat mammary epithelium, centrosome morphology

remained unchanged (Figs 1-5,7-10). Each centrosome was composed of a cylindrical

centriole and surrounding percentriolar matrix (Figs 2-5, 8—10). Centrioles in trans-

verse section were 200-220 (x = 210) nm in diameter and exhibited the typical 9 x 3

'pinwheel' configuration of microtubules (Figs 4, 8). In longitudinal section,

centrioles were 330-380 (x = 360) nm in length (Figs 2, 3, 9, 10). The pericentriolar

matrix was homogeneous and electron-dense (Figs 3, 5, 8—10).

Through serial thin-section analysis, it was determined that 32 of 33 non-mitotic

epithelial cells contained a single centriole, which was located in the apical region of

Figs 1 to 4. Centrioles in virgin (Figs 1, 2) and 14-day pregnant (Figs 3, 4) animals.Fig. 1. Survey micrograph of epithelial cell showing centriole (arrow) in longitudinal

section. Note that the centriole is located in the apical region of the cell; n, nucleus.X10000; bar, 2-0^m.

Fig. 2. High magnification of centriole shown in Fig. 1. The centriole (arrow) isembedded in a homogeneous pericentriolar matrix. X41 000; bar, 0-25^m.

Fig. 3. Longitudinal section of centriole (arrow) near apical plasma membrane(arrowheads). Note that the long axis of the centriole is almost parallel to the plasmamembrane; alveolar lumen (/), and cytoplasmic lipid droplet (eld). X47 000; bar, 0-5 /.tm.

Fig. 4. Centriole (arrow) in transverse section showing typical 9 x 3 pinwheel con-figuration of microtubules. The centriole is located near the apical plasma membrane(arrowheads); alveolar lumen (/). X92000; bar, 0-1 fan.

Centrioles in mammary gland

Figs 1-4

188 D. P. Dylewski and T. W. Keenan

the cell (Figs 1-4, 7-10). In one cell two centrioles were located near the apical plasmamembrane (not shown in figures). The centrioles in cells from virgin rats werepositioned equidistant between the nucleus and the apical plasma membrane (Figs 1,2). In all other developmental stages, however, centrioles were located within 520 nmof the apical plasma membrane (Figs 3, 4, 7—10). The orientation of the centriole tothe apical plasma membrane was variable, except during pregnancy when the long axisof the centriole was nearly parallel to the surface of the membrane (Figs 3, 4, 7, 8).

During the first two weeks of pregnancy, mitotic figures were observed frequentlywithin epithelial cells (Figs 5,6). The mitotic spindle was composed of a diplosomeat each nuclear pole, and numerous chromosomal microtubules (Fig. 5). Eachdiplosome consisted of two centrioles that were oriented at right angles (90°) to eachother (Fig. 5). At the completion of mitosis a single diplosome was associated witheach interphase nucleus (not shown in figures). Because most postmitotic cells (count33) contained only a single centriole it was assumed that one of the two diplosomalcentrioles had disintegrated. The postmitotic disintegration of centrioles is a well-documented phenomenon known to occur in both plant and animal cells (for review,see Wheatley, 1982).

In the premitotic cells of virgin animals, centrioles were located in the apical regionand not at the nuclear poles (Figs 1,2). During mitosis in pregnant animals pairedcentrioles were positioned at each spindle pole in close association with numerousspindle microtubules (Fig. 5). In postmitotic cells of pregnant, lactating and involut-ing animals, centrioles were positioned again in the apical region and not at the nuclearpoles (Figs 7-10). This intracellular repositioning of centrioles during epithelial celldevelopment appears to be triggered by the process of nuclear division, and could beaccomplished by migration or disintegration and reassembly of centrioles. Two linesof evidence support the possibility of centriole migration. First, centrioles were obser-ved along the presumed migratory pathway between apical and nuclear positions.Second, centrioles in stages of disintegration and reassembly were never observed.Although centriole migration appeared to be a naturally occurring phenomenon ofepithelial cell development, the mechanisms for control and coordination by the celland the role of microtubules in this process remain unknown.

The migration of centrioles has been documented during the process of differentia-tion in many plant and animal cells (for review, see Wheatley, 1982). Perhaps the bestexample is in the olfactory sense organs of mammals (Heist & Mulvaney, 1968;Mulvaney & Heist, 1971), in which nuclear-associated centrioles of the columnarepithelium migrate apically for a distance of five times that of the diameter of the rrlain

Figs 5, 6. Mitotic spindle apparatus of 7-day pregnant animals.Fig. 5. Polar region of mitotic figure showing paired centrioles (arrows) arranged at

right angles (90°) to each other; microtubules (arrowheads), and lateral plasma mem-branes (pm). X41000; bar, 0-5/xm.

Fig. 6. Survey micrograph of epithelial cell showing metaphase chromatin plate (largearrows) in oblique section. Note that the cell does not exhibit polarity. Secretory vesicles(small arrows) are distributed throughout the apical (a), medial (m) and basal (b) regionsof the cell, and mitochrondria (mt) show an enrichment at the cell periphery. X9000; bar,2-0/an.

Centrioles in mammary gland 189

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Figs 5 and 6

D. P. Dylewski and T. W. Keenan

8Figs 7 and 8

Centrioles in mammary gland

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Figs 9, 10. Centrioles in mammary epithelial cells of 7-day lactating (Fig. 9), and 4-dayinvoluting (Fig. 10) animals.

Fig. 9. Centriole (arrow) in longitudinal section and pericentriolar matrix: apical plas-ma membrane (arrowheads), portion of milk lipid globule (mlg), and casein micelles (me)in alveolar lumen (/). X50000; bar, 0 5 ^m.

Fig. 10. Longitudinal section of centriole (arrow) located near apical plasma mem-brane. Note that the alveolar lumen (/) contains a finely fibrillar and granular material.X41000; bar, 0-

cell body, and eventually form sensory cilia. In mammary epithelial cells centriolesmigrate half the length of the cell body (x = 8-0/im). Examples of the intercellularmigration of centrioles are not uncommon. This capacity was demonstrated in thenurse cells of the oocyte in Drosophila, in which centrioles passed from the former to

Figs 7, 8. Centrioles in epithelial cells of 21-day pregnant animals.Fig. 7. Survey micrograph of epithelial cell showing polar arrangement of organelles

and secretory vesicles; basal region (b), rough endoplasmic reticulum (er); medial region(m), Golgi apparatus (G), nucleus (n); apical region (a), secretory vesicles (small arrows),and centriole (large arrow). Mitochondria and cytoplasmic lipid droplets (eld) aredistributed evenly throughout the cell. Note that the centriole (large arrow) is located nearthe apical plasma membrane (arrowheads); alveolar lumen (/). X8000; bar, 2-0/im.

Fig. 8. Centriole (arrow) in transverse section near apical plasma membrane(arrowheads); alveolar lumen (/). X41000; bar, 0-5/mi.

192 D. P. Dylewski and T. W. Keenan

the latter during egg development (Mahawald & Strassheim, 1970; Mahowald, Caul-ton, Edwards & Floyd, 1979).

The determination of polarity is of enormous importance in all cells, particularlyepithelial cells, in which the movement of secretory products is unidirectional.Although numerous theories have been proposed concerning the role of centrioles indetermining cell polarity (Bornens, 1978; Albrecht-Buehler, 1977, 1979), there is noconclusive evidence to support such hypotheses. From our observations we can statewith certainty that mammary epithelial cells exhibit polarity in all developmentalstages when the centriole is located in the apical region. The polarized arrangement ofepithelial cell organelles and secretory products was first detectable on day 6 or day 7 ofpregnancy (total 22-day gestation period) and became more obvious in later develop-mental stages when cellular organelles increased in size and complexity. Each polarizedcell could be divided into three regions (e.g. apical, medial and basal) based on thecomposition of cytoplasmic structures (Fig. 7). Rough endoplasmic reticulum was thepredominant organelle in the basal region of the cell (Fig. 7). The nucleus and Golgiapparatus were usually present in the medial region (Fig. 7). Secretory vesicles, a singlecentriole and frequently a portion of the Golgi apparatus were found in the apical region(Fig. 7). Mitochondria and cytoplasmic lipid droplets were distributed throughout thecell (Fig. 7). Epithelial cell polarity was most obvious during lactation when organelledevelopment was complete, and secretion of milk serum and lipid was at a maximum.

During mitosis epithelial cells were typically apolar with respect to the intracellulardistribution of organelles and secretory products (Fig. 6). In cells containing mitoticfigures condensed chromatin was found in the central region and all organelles andcytoplasmic structure were observed on the periphery of the cell. The presence ofsecretory vesicles in the basal region of the cell was the clearest indication that the celllacked polarity (Fig. 6).

In conclusion, apically positioned centrioles were found in polarized cells, andnuclear-associated centrioles were found in apolar cells. Such indirect evidencemight lead one to speculate that the intracellular position of the centriole is theprimary determinant in cell polarity. However, other interpretations of centriolefunction and cell polarity are possible. Clearly, the centriole will remain an enigmaand its function uncertain until they are studied in greater detail at the cellular andmolecular levels.

This research was supported by grants GM31244 from the National Institute of General MedicalScience and PCM82-41913 from the National Science Foundation.

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(Received 25 April 1984 -Accepted 26 June 1984)