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Mechanical stimulation of mammary gland development in virgin and pregnant rats
tlORSIONI~S
AND Il1-11AVIOR
7, 183-197 (1976)
Mechanical Stimulation of Mammary Gland Development in Virgin and Pregnant Rats LORRAINE l3ychokqy
ROTH I-IERKENKOHL
Departtnetlt,
Temple
University,
and CAROL CAMPBELL PhiladeIphia.
Pennsylvania
I9122
‘I he mammary glands of pregnant Sprague-Dawley rats prevented from self-licking by collars around the neck (n = 9) were only about 50% as dcvcloped as those of pregnant rats without collars (n = 9). Howcvcr. when the ventral body surface of pregnant collared lats was slim&ted mechanically 01 = 9), mammary glands increased in size, underwent lobuloalveolar growth, and secreted milk in a manner similar to normal pregnant rats. When virgin rats were stimulated mechanically for 22 days (n = 9), their mammary glands had significantly greater secretury tissue and lobuloalveolar development than did the glands of nonstimulated virgin rats (n = 9). Stimulated rats were tied to the stimulating device under ether anesthesia. Because cthcr and restraint have been shown under some conditions to be strcssors that increase development of the mammary glands, two additional pregnant collared control groups were added. One was restrained on the device without stimulation (n = 9). The other was lightly anesthetized with ether (n - 10). Mammary undcrdcvclopmcnt occurred in both groups. This suggested that stress of ether and restraint were unlikely causes of the mammary development in the slimulated group.
Suckling
and
other
stimulation
of
the
ventral
body
surface
have
long
been known to maintain lactation and cause mammary growth in a variety oi postparturient mammals, including the rat (see reviews by Cowie and t’olley, 1961; Cross, 1967). However, only in more recent years has the influence of sensory stimulation before parturition been examined. Roth and Rosenblatt (1966, 1968) prevented pregnant rats from self-licking by collars around the neck (Birch, 1956) and found at the end of ge5TaTiOn That the mammary glands of those rats were only about half as developed as those of control animals. The glands contained fewer secretory cells and had little or no milk. On this basis, they concluded that self-licking was a stimulus for mammary development during gestation, at a time when mammary glands were previously thought to he only under endogenous hormonal control. I.ater studies confirmed that report and indicated further that prevention of self-licking during gestation decreased DNA activity in the mammary glands (McMurtry and Anderson, 197 1; Whitworth, 1972). 183 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
184
IIICRRENKOIIL AND ('AMPBIC1.L
There is no evidence that sensory stimulation causes development of the mammary glands in virgin rats. In a classic paper, Selye and McKeown (I 934) reported that suckling young induced pseudopregnancy in formerly pregnant rats. However, attempts to induce pseudopregnancy in virgin rats were to no avail. “Their nipples. . . [were tool small . . ” and “. . . active suckling occurred only in one case” (p. 685). During the reproductive cycle there is usually no need for virgin rats to have enlarged mammary glands: They are not fostering suckling young. However the growing body of evidence that virgin animals can be maternal under certain conditions (Rheingold, 1963) encouraged studies of the conditions that in turn might foster mammary growth and secretion. Thus in the present experiment we examined two questions: (1) does mechanical stimulation of the ventral surface of pregnant rats prevented from self-licking increase the otherwise underdeveloped state of the mammary glands, and (2) can sensory stimulation promote development of the mammary glands in virgin rats. METHODS Animals Sixty-four female Sprague-Dawley rats were obtained from the Camm Research Institute (Wayne, New Jersey) at 200-250 g, about 2 months of age. Forty-six of the females were mated on the day of estrus as determined by vaginal smears. The 18 females remaining constituted the virgin groups. All animals were housed individually in 22 X 29 X 13 cm Fibreglas cages with San-i-ccl bedding. Because females with collars cannot lift their necks, they were fed Purina pellets and water ad lib. in glass jars on the floor, instead of the usual wall containers. They were kept in colony rooms maintained at a temperature of 72°F on 16 hr of light daily beginning at 10 AM. Experimental Design Pregnant animals were randomly divided into five groups as follows: Group 1, the pregnant collared stimulated animals (n = 9) wore collars to prevent self-licking but also received mechanical stimulation on the machine; Group 2, the pregnant collared machine animals (n = 9) wore collars but were placed on the machine without stimulation; Group 3, the pregnant collared animals (n = 9) only wore collars; Group 4, pregnant collar etherized rats (n = lo), wore collars but received only ether, previously used to anesthetize the other animals as they were placed on the machine; and finally, Group 5, a normal group of pregnant rats (n = 9).
MKIIAXICAL
STIMUI.ATION
OF RAT MAMMARY
185
GLANDS
Virgin estrus-cycling females were divided into two groups. One group. the virgin stimulated animals (11 = 9), were mechanically stimulated on the machine for 22 days. The second group, the control virgins (12= 9), remained untreated. Ether and restraint have been shown in the past under certain conditions to be stressors that induce development of the mammary glands (Meites, Nicoli, and Talwalker, 1963; Grosvenor, Krulick, and McCann, 1968). Among the stress effects typically found during gestation are abortion or absorption of fetuses, increased adrenal gland weight and low birth weights of surviving young (Selye, 1950; Grdnroos, Kappila and Soiva, 1963; Weir and Defries, 1963). Therefore, to determine whether stress and restraint associated with mechanical stimulation might have led to development of the mammary glands, we killed the females at the end of the study and recorded body and adrenal gland weight. in the case of the pregnant females, we also counted the number of fetuses in the uterine horns, As shown in Table 1, none of the usua! stress effects were apparent. Despite differences in treatment, most of the pregnant females weighed about 250 g and, on the average, they carried 10 or 11 fully-developed pups. Also, their adrenal gland weights were within normai range.
~xperirnmtal Procedures Animals wore collars made of l/3 cm U.S. brand gasket rubber, obtained from plumbing supplies. The collars were cut into the shape of a doughnut with the outer diameter being 9 cm. The hole in the center was cut to TAHLI:
1
Effects of Treatments on the Body Weights of the Females, as well as Their Adrenal Gland Weights and the Number of Fetuses
-
Treatment .-.-.-.-
Pregnant collared stimulated Pregnant Pregnant collared machine Pregnant collared etherized Pregnant collared Virgin stimulated Virgin ‘W’ith litter.
Body wt. of female in gram+ B (X ? Sk,? --.-.----._.._._
Adrenal &kind wt. in grams (X T SE) _ _ _-__. __.._ -_--
Number of fetuses (X f A-It? -.-..-
9 9
250 f 21.22 254 i 21.45
.64 +_.07 .66 7 .oo
10.0 5 1.90 10.3 T 2.62
9
242 A 31.42
.68 + .06
10.0 i 2.14
252? 248 I 203 ! 212 j
.65 .68 .63 .59
11.8 i 1.04 10.0 ‘* 1.95
10 9 9 9
17.61 14.21 14.72 11.19
i .06 zt .04 1 .27 f .05
186
III‘RREh’KOHL
AND CAMPBJ~.LL
fit the individual neck and lined with soft felt to prevent irritation. A slit through each collar was made so it could be spread open and slipped around the neck. Wire was then fastened through four holes on each side of the slit to keep the collar closed. Mechanical stimulation was delivered to the ventral surface of animals by means of a mechanical stimulator device (Fig. IA, B). Animals were lightly anesthetized with ether and placed on top of the Plexiglas platform and restrained with ties around each leg. Stimulation was then accomplished under ether by the rotation of two large cylinders lined with sable-hair brushes at the rate of 32 rpm for 30 min daily for 22 days. At the end of the study a l-cm square sample of mammary tissue was obtained from the inguinal mammary gland and prepared histologically according to procedures previously reviewed (Roth and Rosenblatt, 1968). It was serially sectioned at 5 ym intervals perpendicular to the plane of the skin. Fifteen consecutive sections from each sample were mounted, five to a slide, and stained with Harris’ haematoxylin and eosin. Each gland sample was then assessedfor its degree of development by objective measures of the amount of secretory tissue, as well as by subjective ratings (Benson and Folley, 1957; Roth and Rosenblatt, 1966, 1968). One section was randomly chosen and measured objectively for the “percentage of secretory tissue,” or the amount of secretory tissue as a proportion of the total amount of glandular tissue. Subjectively, it was rated for “L-A developmen t,” or the stage of development of the lobuloavealar system on a O-3 scale, as well as for “S-activity,” the amount of secretory tissue present, on a l-3 scale. L-A development was rated as follows: 0 = alveoli absent or rare; 1 = few alveoli, lobes distinct but small and scattered; 2 = alveoli nearly filling each lobe, lobes large and compact; 3 = alveoli densely packed, filling every one of the lobes which are larger and more compact than in 2. S-activity received the following scores: 1 = secretion absent or rare; 2 = secretion sparse but present in most of the alveoli; 3 = secretion filling nearly all the alveoli and ducts throughout the tissue. The “percentage of secretory tissue” was obtained by magnifying the image of the section 40X and projecting it at a fixed distance with a Bausch and Lomb microprojector onto a sheet of paper of standard weight (Oshima and Goto, 1955; Squartini, 1957). The outlines of both the secretory and nonsecretory tissue were drawn, cut out, and weighed. The “percentage of secretory tissue” was then calculated as a proportion of the total mass, and expressed as percentage. RESULTS Figure 2 gives the mean percentage secretory tissue in the mammary glands of stimulated and nonstimulated virgin and pregnant rats. A one-way
MECHANICAL STIMULATION
OF RAT MAMMARY GL4NDS
187
Fig. 1A. Top view of the mechanical stimulating machine, revealing the 10 X 9 cm cutout through which the ventral body surface of the rat, tied to the platform above, is stroked by two rows of #2 sable-hair brushes on the 36 X 46 cm Plexiglas platform below.
Fig. 11s. Side view of the mechanical stimulating machine. Two Plexiglas cylinders appearing white in the center of the picture are lined with sable-hair brushes which are seen here protruding through the opening in the platform above. To the left center (7) is a cervical probe, not used in this study, which when positioned in place can also be mechanically driven to stimulate the genital region of the female rat.
188
HERRENKOIIL
ASD CAMPBELL
analysis of variance was significant [F(6,57) = 4.86; p < .Ol] . Mean comparisons assessedby the Duncan’s Multiple Range Test showed that the percentage secretory tissue did not differ significantly between the pregnant collared stimulated rats and the normally pregnant rats (81 and SS~?Jsecretory tissue respectively, p > .05). However, both these groups had significantly greater mean percentage secretory tissue than the three respective control groups (the pregnant collared machine, pregnant collared etherized and pregnant collared rats; 63, 58 and 53%, respectively, all 8 ps< .05). Furthermore, the stimulated virgin group had significantly greater secretory tissue than its control group, the nonstimulated virgin rats (30 and 1l%, respectively, p < .05). The only other significant differences occurred when pregnant collared stimulated and pregnant collared groups were compared with virgin groups (all four ps < .Ol). Figure 3 gives the mean ratings for secretory activity (S-A) and lobuloalveolar development (L-A) in stimulated and nonstimulated virgin and pregnant rats. Again it can be seen that the mammary glands of the pregnant collared stimulated rats strikingly resembled those of normally pregnant rats. Mean S-A and LA ratings in the pregnant collared stimulated group approached those of the normally pregnant rats (being 2.2 and 2.4, respectively, in the first case; whereas, in the latter group the values were 3.0 and 3.0). In both groups, the lobules were large and compact and almost all the aveoli MAMMARY AND
N
GLAND VIRGIN
DEVELOPMENT ANIMALS
IN PREGNANT t
SE.)
9
PREGNANT
9
PREGNANT COLLARED MACHINE
IO
PREGNANT COLLARED ETHERISED
9
PREGNANT COLLARED
9
VIRGIN STIMULATED
% SECRETORY
TISSUE
Pig. 2. The percentage of secretory stimulated
(MEAN
and nonstimulated
TO
TOTAL
MAMMARY
GLAND
tissue to total mammary gland virgin and pregnant rats (mean + Sk?.
tissue in
MECHANICAL STlMULATlON MAMMARY
GLAND
AND
N
01: RAT MAMMARY GLANDS
DEVELOPMENT
UNSTIMULATED
RATS
IN (MEAN
189
STIMULATED + S.E
)
9
PREGNANT COLLARED MAC”lNE
IO
PREGNANT COLLARED ETHERISLD
PREGNANT COLLARED
VIRGIN STIMULATED
9
5
0 L-ANO Ai.VEOl.1
L-A S-A
IO FEW ABSENT
15
2.0 L-A. S-A
2.5
MANY IN MAJORITY OF CELLS
1
30 L-A S-A
ALVEOLI FILL l.OBULES SECRETION IN ALL ALVEOLI
LEGEND S-A
B.j
L-A
0
Fig. 3. Ratings of secretory activity (S-A) and lobulo-alveolar development (L-.4) in the mammary glands of stimulated and nonstimulated virgin and pregnant rats (mean GK). L-A development was rated as follows: 0 = alveoli absent or rare; 1 = few alveoli, lobes distinct but small and scattered; 2 = alveoli nearly filling each lobe, large and compact; 3 = alveoli densely packed, filling every one of the lobes which are larger and more compact than in 2. S-A was rated as follows: 1 = secretion absent or rare; 2 = secretion sparse but present in most of the alveoli; 3 = secretion filling nearly all the alveoli and ducts throughout the tissue.
contained milk (Figs. 4A, B). None of the pregnant control groups had mammary glands that large (mean S-A and L-A for the pregnant collared machine, pregnant collared etherized and pregnant collared rats being 1.6 and 1.9, 2.0 and 2.0, and 1.4 and 1.7, respectively). The mammary glands in those groups either contained small lobes scattered throughout the tissue where secretions were rare (Fig. 4C, D) or else they contained many lobes, with only some of the alveoli being large and with milk (Fig. 4E). It can also be seen in Fig. 3 that stimulated virgins had greater mammary development than nonstimulated virgins (mean ratings S-A and L-A in the latter being 0 and 0; in the former, 1.0 and 1.2). Whereas the mammary glands of untreated virgins characteristically contained only an atrophic duct system embedded in a connective mass (Fig. 5A), the mammary glands of stimulated virgins contained ducts that had begun to differentiate into alveoli and lobes, and secretion began to be present (Fig. 5B).
190
HERRENKOIIL AND CAMPBELL
Mann-Whitney U tests on mean S-A and L-A ratings revealed that pregnant collared stimulated animals had significantly greater secretory activity and lobuloalveolar development than at least two of its control groups (for the pregnant collared stimulated rats, cls = 64.0 and 58.5, n, = 9, n2 = 9, p < .002: for pregnant collared rats, Us = 62.0 and 45.2; n, = 9, n2 = 9, p < .002). However, they did not have significantly greater mean S-A and L-A ratings than the third control group (for pregnant collared etherized rats, Us = 16.5 and 15.5; nl = 10, nz = 9; p > .05). As expected, mean ratings of S-A and L-A in virgin stimulated animals were significantly greater than those of nonstimulated virgin rats (Us = 19.5 and 20.4; nl = 9; n2 = 9; p < .05). DISCUSSION Pregnant rats prevented from self-licking that received mechanical stimulation of the ventral body surface were observed to have mammary glands almost as well-developed as those of normally pregnant untreated rats. As expected on the basis of past results (Roth and Rosenblatt, 1966, 1968) both kinds of females had significantly greater mammary development than females similarly prevented from self-licking by collars around the neck (Birch, 1956). Moreover, mechanically-stimulated females had significantly greater mammary development than control females that were also prevented from self-licking, but that were either only restrained on the machine or placed under light anesthesia. Thus, by all the measures we employed, whether subjective or objective, mechanical stimulation of the ventral surface of pregnant rats prevented from self-licking was found to increase development of the mammary glands. The only minor and unexpected result was that ratings of glandular condition did not differ significantly between the primary experimental group and the etherized control group, due, most likely, to the larger individual differences in the latter. That stress of mechanical stimulation was the cause of the increase in mammary development appears unlikely for the following reasons. First, we found that neither ether nor restraint induced development of the mammary glands (Figs. 2, 3). Under a number of conditions, both these factors have been found to be stressors that enhance development of the mammary glands (Meites, Nicoll and Talwalker, 1963; Grosvenor, Krulick and McCann, 1968). Second, there were no signs that pregnancy was disturbed. If stress was active we would have expected to find that fetuses were either aborted or absorbed, that maternal adrenal gland weights were high, and that surviving young were underweight (Selye, 1950; Cronroos, Kappila and Soiva, 1963; Weir and Defries, 1963). None of these signs were apparent (Table 1). Thus the most likely explanation is that mechanical stimulation itself, and not secondary effects associated with it, was the cause of the increased development of the mammary glands.
FIGURE
FIGURE
4C
4D
MECHANICAL STIMULATION OF RAT MAMMARY GLANDS
193
That self-licking is an important stimulus for development of the mammary glands during gestation was first demonstrated by Roth and Rosenblatt (1966, 1968) when they prevented pregnant rats from self-licking by collars around the neck and found that mammary development was reduced. In an analysis of biochemical changes in the mammary glands, McMurtry and Anderson (1971) reported that the glands of pregnant rats prevented from self-licking by collars around the neck contained amounts of DNA and RNA that were significantly smaller than those in normally pregnant rats. Moreover, in an extensive study of prevention of self-licking employing a variety of techniques, Whitworth (1972) found that mammary glands were also underdeveloped in pregnant rats prevented from self-licking by selective thelectomy . Again, by all the measures we employed, virgin rats receiving stimulation had significantly greater mammary development than normal virgin rats.
194
HERRENKOHLANDCAMPBELL
MEClIANlCAL STIMULATION OF RAT MAMMARY GLANDS
195
Instead of containing only the atrophic duct system characteristic of virgin rats, stimulated virgins had mammary glands resembling those of the first trimester of gestation (see review by Cowie and Folley, 1961). Although no virgins in this report received restraint without mechanical stimulation, restraint as a factor in mammary growth can be ruled out on the basis that stress effects were not apparent in pregnant rats. Thus, it was shown that mechanical stimulation can induce mammary development in virgin rats, in the absence of gestational states. That mechanical stimulation can induce mammary development in virgin rats provides scientific support for a number of puzzling anecdotal reports. Zaks (1962), for example, claimed that massage of the teats in virgin heifers and goats caused mammary growth and secretion and that when applied to parturient heifers, it facilitated greater milk yield. Moreover, reports through the years have suggested that poultices applied to the teats, as well as rubbing, cause nipple development in “virgin” animals in a variety of mammals, including humans. It is possible, therefore, that virgin girls, animals, and even men, can secrete milk under mechanical means (Seifer, 1921; Selye and McKeown, 1934; Folley and Malpress, 1948; Zaks, 1962; Knott, 1970). Given the observation that mechanical stimulation increased mammary development in both virgin and pregnant rats, there are several questions remaining. One, what hormones are released by sensory stimulation which, in turn, cause growth of the mammary glands? Gonadotrophin secretion from the anterior pituitary gland is the most likely cause, either directly, through prolactin (which causes secretion of milk) and/or indirectly, through stimulation of ovarian hormones which, in turn, produce development of the mammary glands (see review by Cowie and Folley, 1961). One ovarian hormone, progesterone, has now been shown to be the response in a neuroendocrine reflex involving copulatory stimulation of the vaginocervical region in female rats (Dilley and Adler, 1968; Adler, 1969). Although prolactin seems the most likely choice, (because of its primary action on mammary gland cells), measurement of prolactin in the pituitary gland have not as yet revealed systematic changes as a function of self-licking (Grosvenor and Mena, 1969). Serum assays of prolactin involving more refiied techniques need to be done. An equally interesting question is the relationship between self-licking during gestation and subsequent maternal behavior. In 1956, Birch devoted only a few paragraphs in a much-cited theoretical paper to a description of an experiment he performed. He raised female rats with collars from weaning and found that at birth they ate 9% of their young, and that, failing to assume an appropriate nursing posture over the surviving young, the mothers allowed the young to die. Christopherson and Wagman (1965) have repeated the study in part, but instead of infant mortality, they found a less dramatic result: prevention of self-licking led only to a significant reduction in pup
196
HERRENKOIIL
ASD CAMPBELL
weight. These observations, in conjunction with the present report, suggest that one way prevention of self-licking during gestation can alter subsequent development of the young is by means of a lactational dysfunction associated with underdeveloped mammary glands.
ACKNOWLEDGMENTS We gnatefully acknowledge support for this research from a Biomedical Sciences Support Grant to Temple University and by National Institute of Child Health and Human Development Research Grant No. HD-04715 awarded to Lorraine Roth tlerrenkohl as principal investigator.
REFERENCES Adler, N. T. (1969). Effects of the male’s copulatory behavior on successful pregnancy of the female rat. J. Comp. Physiol. Psychol. 69, 613622. Benson, G. K., and Folley, S. J. (1957). The effect of oxytocin on mammary gland involution in the rat. J. Endocrinol. 16, 189-201. Birch, H. G. (1956). Sources of order in the maternal behavior of animals. Amer. J. Orthopsychiat.
26, 270-284.
Christophcrson, L:.. R., and Wagman, W. (1965). Maternal behavior in the albino rat as a function of self-licking deprivation. J. Camp. Physiol. qYscho2. 26, 270-294. Cowie, A. T., Folley, S. J. (1961). The mammary gland and lactation. In W. C. Young (Ed.), Sex and Internal Secretions, 3rd ed. Williams and Wilkins, Baltimore, MD. Cross, B. A. (1967). Neural control of oxytocin secretion. In L. Martini and W. F. Ganong (Ms.), Neuroendocrinology. Academic Press, New York. Dilley, W. G., and Adler, N. T. (I 968). Postcopulatory mammary gland secretion in rats. Sot. Exp. Biol. Med. 129, 964-966.
Folley, S. J., and Malpress, P. H. (1948). Hormonal control of mammary growth. In G. Pincus and K. V. Thimann (Fds.), 7he Hormones. Academic Press, New York. Gronroos, M., Kauppila, O., and Soiva, K. (1961). Changes in the vaginal smears of rats during normal and disturbed pregnancies. Actu Endocrinol. 37, 309-314. Grosvenor, C. E. (1965). lvidence that exteroceptice stimuli can release prolactin from the pituitary gland of the lactating rat. Endocrinology 76, 340-342. Grosvenor, C. E., Krulich, L. and McC~M, S. (1968). Depletion of pituitary concentration of growth hormone as a result of suckling in the lactating rat. Endocrinology 82, 617619. Grosvenor, C. E., and Mena, F. (1969). Failure of self-licking of nipples to alter pituitary prolactin concentration in lactating rats Horm. Behov. I, 85-91. Knott, J. C. (1907). Abnormal lactation in the virgin; in the old woman; in the male; in the newborn of either sex (“witches milk”). Amer. Med. 2, 373. Lehrman, D. S. (1961). Hormonal regulation of parental behavior in birds and infrahuman mammals. In W. C. Young (Ed.). Sex and Internal Secretions, 3rd ed. Williams and Wilkins, Baltimore, MD. McMurtry, J. P. and Anderson, R. R., (1971). Prevention of self-licking on mammary gland development in pregnant rats. Proc. Sot. Exp. Bio. Med. 137, 354-356. Meites, J. NicoII, C. S., and TaIwaIker, P. K. (1963). The central nervous system and the secretion and release of prolactin. In A. V. Nalbandov (Ed.), Advances in Neuroendocrinolofl, University of Illinois Press, Urbana, IL.
MECHANICAL STIMULATION
OF RAT MAMMARY GLANDS
197
Oshima, %l., and Goto, T. (1955). A study of involution of mammary gland after weaning in the albino raL. Bull. :l;atr~ host. Agric. Sci., Tokyo (Ser. G), 11, 81-87. Rheingold, 11. (Ed.). (1963). Maferrlal Behavior in Mammals, John Wiley and Sons, New York. Kosenblatt, J. S., and Lehrmdn, D. S. (1963). Maternal behavior of the laboratory rat. In II. Rheingold (Ed.), Marernal Behavior in Mammals. John Wiley and Sons, New; York. Roth, 1.. L., and Rosenhla~t, J. S. (1966). Mammar) r glands of pregnant rats: Development stimulated by licking. Science 151, 1403-1404. Roth, L. L., and Rosenblatt, J. S. (1967). Changes in self-licking during pregnancy in the rat. J. Cbrnp. PhysioL Psychol. 63, 397-400. Roth, L. I.., and Rosenhlatt, 1. S. (1968). Self-licking and mammary development during pregnancy in the rat. .I. b,Wocrinol. 42, 363-378. Seifert, M. J. (1920). Abnormal lactation. J. Anzer. !Med. Assoc. 74, 1634. Selye, Hans (1950). Stress. Acta Incorporated, biontreal, Canada. Selye, II., and YcKeown, T. (1934). Production of pseudo-pregnancy by mechanical stimulation of the nipples. Froc. Sot. Exp. Biol. 31, 638-687. Squartini, t;. (1957). A new method for the study of the mammary gland of the mouse. Cancer 10, 179-182. Weir, M. W., and De I:rics, J. C. (1963). Blocking of pregnancy in mice as a function of stress. Ps~~clzol.Repts. 13, 365-366. Whitworth, Neil (1972). Relationships between patterns of grooming, endocrine function and mammary gland development in the pregnant rat. Doctoral dissertation, submitted to Rutgers University, New Brunswick, NJ. Zaks, M. G. (1962). ‘Uze Motor Apparatus of the Mammary Gland. Translated by D. G. Fry., A. T. Cowie (Ed.). Oliver and Hoyd, London.
AND Il1-11AVIOR
7, 183-197 (1976)
Mechanical Stimulation of Mammary Gland Development in Virgin and Pregnant Rats LORRAINE l3ychokqy
ROTH I-IERKENKOHL
Departtnetlt,
Temple
University,
and CAROL CAMPBELL PhiladeIphia.
Pennsylvania
I9122
‘I he mammary glands of pregnant Sprague-Dawley rats prevented from self-licking by collars around the neck (n = 9) were only about 50% as dcvcloped as those of pregnant rats without collars (n = 9). Howcvcr. when the ventral body surface of pregnant collared lats was slim&ted mechanically 01 = 9), mammary glands increased in size, underwent lobuloalveolar growth, and secreted milk in a manner similar to normal pregnant rats. When virgin rats were stimulated mechanically for 22 days (n = 9), their mammary glands had significantly greater secretury tissue and lobuloalveolar development than did the glands of nonstimulated virgin rats (n = 9). Stimulated rats were tied to the stimulating device under ether anesthesia. Because cthcr and restraint have been shown under some conditions to be strcssors that increase development of the mammary glands, two additional pregnant collared control groups were added. One was restrained on the device without stimulation (n = 9). The other was lightly anesthetized with ether (n - 10). Mammary undcrdcvclopmcnt occurred in both groups. This suggested that stress of ether and restraint were unlikely causes of the mammary development in the slimulated group.
Suckling
and
other
stimulation
of
the
ventral
body
surface
have
long
been known to maintain lactation and cause mammary growth in a variety oi postparturient mammals, including the rat (see reviews by Cowie and t’olley, 1961; Cross, 1967). However, only in more recent years has the influence of sensory stimulation before parturition been examined. Roth and Rosenblatt (1966, 1968) prevented pregnant rats from self-licking by collars around the neck (Birch, 1956) and found at the end of ge5TaTiOn That the mammary glands of those rats were only about half as developed as those of control animals. The glands contained fewer secretory cells and had little or no milk. On this basis, they concluded that self-licking was a stimulus for mammary development during gestation, at a time when mammary glands were previously thought to he only under endogenous hormonal control. I.ater studies confirmed that report and indicated further that prevention of self-licking during gestation decreased DNA activity in the mammary glands (McMurtry and Anderson, 197 1; Whitworth, 1972). 183 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
184
IIICRRENKOIIL AND ('AMPBIC1.L
There is no evidence that sensory stimulation causes development of the mammary glands in virgin rats. In a classic paper, Selye and McKeown (I 934) reported that suckling young induced pseudopregnancy in formerly pregnant rats. However, attempts to induce pseudopregnancy in virgin rats were to no avail. “Their nipples. . . [were tool small . . ” and “. . . active suckling occurred only in one case” (p. 685). During the reproductive cycle there is usually no need for virgin rats to have enlarged mammary glands: They are not fostering suckling young. However the growing body of evidence that virgin animals can be maternal under certain conditions (Rheingold, 1963) encouraged studies of the conditions that in turn might foster mammary growth and secretion. Thus in the present experiment we examined two questions: (1) does mechanical stimulation of the ventral surface of pregnant rats prevented from self-licking increase the otherwise underdeveloped state of the mammary glands, and (2) can sensory stimulation promote development of the mammary glands in virgin rats. METHODS Animals Sixty-four female Sprague-Dawley rats were obtained from the Camm Research Institute (Wayne, New Jersey) at 200-250 g, about 2 months of age. Forty-six of the females were mated on the day of estrus as determined by vaginal smears. The 18 females remaining constituted the virgin groups. All animals were housed individually in 22 X 29 X 13 cm Fibreglas cages with San-i-ccl bedding. Because females with collars cannot lift their necks, they were fed Purina pellets and water ad lib. in glass jars on the floor, instead of the usual wall containers. They were kept in colony rooms maintained at a temperature of 72°F on 16 hr of light daily beginning at 10 AM. Experimental Design Pregnant animals were randomly divided into five groups as follows: Group 1, the pregnant collared stimulated animals (n = 9) wore collars to prevent self-licking but also received mechanical stimulation on the machine; Group 2, the pregnant collared machine animals (n = 9) wore collars but were placed on the machine without stimulation; Group 3, the pregnant collared animals (n = 9) only wore collars; Group 4, pregnant collar etherized rats (n = lo), wore collars but received only ether, previously used to anesthetize the other animals as they were placed on the machine; and finally, Group 5, a normal group of pregnant rats (n = 9).
MKIIAXICAL
STIMUI.ATION
OF RAT MAMMARY
185
GLANDS
Virgin estrus-cycling females were divided into two groups. One group. the virgin stimulated animals (11 = 9), were mechanically stimulated on the machine for 22 days. The second group, the control virgins (12= 9), remained untreated. Ether and restraint have been shown in the past under certain conditions to be stressors that induce development of the mammary glands (Meites, Nicoli, and Talwalker, 1963; Grosvenor, Krulick, and McCann, 1968). Among the stress effects typically found during gestation are abortion or absorption of fetuses, increased adrenal gland weight and low birth weights of surviving young (Selye, 1950; Grdnroos, Kappila and Soiva, 1963; Weir and Defries, 1963). Therefore, to determine whether stress and restraint associated with mechanical stimulation might have led to development of the mammary glands, we killed the females at the end of the study and recorded body and adrenal gland weight. in the case of the pregnant females, we also counted the number of fetuses in the uterine horns, As shown in Table 1, none of the usua! stress effects were apparent. Despite differences in treatment, most of the pregnant females weighed about 250 g and, on the average, they carried 10 or 11 fully-developed pups. Also, their adrenal gland weights were within normai range.
~xperirnmtal Procedures Animals wore collars made of l/3 cm U.S. brand gasket rubber, obtained from plumbing supplies. The collars were cut into the shape of a doughnut with the outer diameter being 9 cm. The hole in the center was cut to TAHLI:
1
Effects of Treatments on the Body Weights of the Females, as well as Their Adrenal Gland Weights and the Number of Fetuses
-
Treatment .-.-.-.-
Pregnant collared stimulated Pregnant Pregnant collared machine Pregnant collared etherized Pregnant collared Virgin stimulated Virgin ‘W’ith litter.
Body wt. of female in gram+ B (X ? Sk,? --.-.----._.._._
Adrenal &kind wt. in grams (X T SE) _ _ _-__. __.._ -_--
Number of fetuses (X f A-It? -.-..-
9 9
250 f 21.22 254 i 21.45
.64 +_.07 .66 7 .oo
10.0 5 1.90 10.3 T 2.62
9
242 A 31.42
.68 + .06
10.0 i 2.14
252? 248 I 203 ! 212 j
.65 .68 .63 .59
11.8 i 1.04 10.0 ‘* 1.95
10 9 9 9
17.61 14.21 14.72 11.19
i .06 zt .04 1 .27 f .05
186
III‘RREh’KOHL
AND CAMPBJ~.LL
fit the individual neck and lined with soft felt to prevent irritation. A slit through each collar was made so it could be spread open and slipped around the neck. Wire was then fastened through four holes on each side of the slit to keep the collar closed. Mechanical stimulation was delivered to the ventral surface of animals by means of a mechanical stimulator device (Fig. IA, B). Animals were lightly anesthetized with ether and placed on top of the Plexiglas platform and restrained with ties around each leg. Stimulation was then accomplished under ether by the rotation of two large cylinders lined with sable-hair brushes at the rate of 32 rpm for 30 min daily for 22 days. At the end of the study a l-cm square sample of mammary tissue was obtained from the inguinal mammary gland and prepared histologically according to procedures previously reviewed (Roth and Rosenblatt, 1968). It was serially sectioned at 5 ym intervals perpendicular to the plane of the skin. Fifteen consecutive sections from each sample were mounted, five to a slide, and stained with Harris’ haematoxylin and eosin. Each gland sample was then assessedfor its degree of development by objective measures of the amount of secretory tissue, as well as by subjective ratings (Benson and Folley, 1957; Roth and Rosenblatt, 1966, 1968). One section was randomly chosen and measured objectively for the “percentage of secretory tissue,” or the amount of secretory tissue as a proportion of the total amount of glandular tissue. Subjectively, it was rated for “L-A developmen t,” or the stage of development of the lobuloavealar system on a O-3 scale, as well as for “S-activity,” the amount of secretory tissue present, on a l-3 scale. L-A development was rated as follows: 0 = alveoli absent or rare; 1 = few alveoli, lobes distinct but small and scattered; 2 = alveoli nearly filling each lobe, lobes large and compact; 3 = alveoli densely packed, filling every one of the lobes which are larger and more compact than in 2. S-activity received the following scores: 1 = secretion absent or rare; 2 = secretion sparse but present in most of the alveoli; 3 = secretion filling nearly all the alveoli and ducts throughout the tissue. The “percentage of secretory tissue” was obtained by magnifying the image of the section 40X and projecting it at a fixed distance with a Bausch and Lomb microprojector onto a sheet of paper of standard weight (Oshima and Goto, 1955; Squartini, 1957). The outlines of both the secretory and nonsecretory tissue were drawn, cut out, and weighed. The “percentage of secretory tissue” was then calculated as a proportion of the total mass, and expressed as percentage. RESULTS Figure 2 gives the mean percentage secretory tissue in the mammary glands of stimulated and nonstimulated virgin and pregnant rats. A one-way
MECHANICAL STIMULATION
OF RAT MAMMARY GL4NDS
187
Fig. 1A. Top view of the mechanical stimulating machine, revealing the 10 X 9 cm cutout through which the ventral body surface of the rat, tied to the platform above, is stroked by two rows of #2 sable-hair brushes on the 36 X 46 cm Plexiglas platform below.
Fig. 11s. Side view of the mechanical stimulating machine. Two Plexiglas cylinders appearing white in the center of the picture are lined with sable-hair brushes which are seen here protruding through the opening in the platform above. To the left center (7) is a cervical probe, not used in this study, which when positioned in place can also be mechanically driven to stimulate the genital region of the female rat.
188
HERRENKOIIL
ASD CAMPBELL
analysis of variance was significant [F(6,57) = 4.86; p < .Ol] . Mean comparisons assessedby the Duncan’s Multiple Range Test showed that the percentage secretory tissue did not differ significantly between the pregnant collared stimulated rats and the normally pregnant rats (81 and SS~?Jsecretory tissue respectively, p > .05). However, both these groups had significantly greater mean percentage secretory tissue than the three respective control groups (the pregnant collared machine, pregnant collared etherized and pregnant collared rats; 63, 58 and 53%, respectively, all 8 ps< .05). Furthermore, the stimulated virgin group had significantly greater secretory tissue than its control group, the nonstimulated virgin rats (30 and 1l%, respectively, p < .05). The only other significant differences occurred when pregnant collared stimulated and pregnant collared groups were compared with virgin groups (all four ps < .Ol). Figure 3 gives the mean ratings for secretory activity (S-A) and lobuloalveolar development (L-A) in stimulated and nonstimulated virgin and pregnant rats. Again it can be seen that the mammary glands of the pregnant collared stimulated rats strikingly resembled those of normally pregnant rats. Mean S-A and LA ratings in the pregnant collared stimulated group approached those of the normally pregnant rats (being 2.2 and 2.4, respectively, in the first case; whereas, in the latter group the values were 3.0 and 3.0). In both groups, the lobules were large and compact and almost all the aveoli MAMMARY AND
N
GLAND VIRGIN
DEVELOPMENT ANIMALS
IN PREGNANT t
SE.)
9
PREGNANT
9
PREGNANT COLLARED MACHINE
IO
PREGNANT COLLARED ETHERISED
9
PREGNANT COLLARED
9
VIRGIN STIMULATED
% SECRETORY
TISSUE
Pig. 2. The percentage of secretory stimulated
(MEAN
and nonstimulated
TO
TOTAL
MAMMARY
GLAND
tissue to total mammary gland virgin and pregnant rats (mean + Sk?.
tissue in
MECHANICAL STlMULATlON MAMMARY
GLAND
AND
N
01: RAT MAMMARY GLANDS
DEVELOPMENT
UNSTIMULATED
RATS
IN (MEAN
189
STIMULATED + S.E
)
9
PREGNANT COLLARED MAC”lNE
IO
PREGNANT COLLARED ETHERISLD
PREGNANT COLLARED
VIRGIN STIMULATED
9
5
0 L-ANO Ai.VEOl.1
L-A S-A
IO FEW ABSENT
15
2.0 L-A. S-A
2.5
MANY IN MAJORITY OF CELLS
1
30 L-A S-A
ALVEOLI FILL l.OBULES SECRETION IN ALL ALVEOLI
LEGEND S-A
B.j
L-A
0
Fig. 3. Ratings of secretory activity (S-A) and lobulo-alveolar development (L-.4) in the mammary glands of stimulated and nonstimulated virgin and pregnant rats (mean GK). L-A development was rated as follows: 0 = alveoli absent or rare; 1 = few alveoli, lobes distinct but small and scattered; 2 = alveoli nearly filling each lobe, large and compact; 3 = alveoli densely packed, filling every one of the lobes which are larger and more compact than in 2. S-A was rated as follows: 1 = secretion absent or rare; 2 = secretion sparse but present in most of the alveoli; 3 = secretion filling nearly all the alveoli and ducts throughout the tissue.
contained milk (Figs. 4A, B). None of the pregnant control groups had mammary glands that large (mean S-A and L-A for the pregnant collared machine, pregnant collared etherized and pregnant collared rats being 1.6 and 1.9, 2.0 and 2.0, and 1.4 and 1.7, respectively). The mammary glands in those groups either contained small lobes scattered throughout the tissue where secretions were rare (Fig. 4C, D) or else they contained many lobes, with only some of the alveoli being large and with milk (Fig. 4E). It can also be seen in Fig. 3 that stimulated virgins had greater mammary development than nonstimulated virgins (mean ratings S-A and L-A in the latter being 0 and 0; in the former, 1.0 and 1.2). Whereas the mammary glands of untreated virgins characteristically contained only an atrophic duct system embedded in a connective mass (Fig. 5A), the mammary glands of stimulated virgins contained ducts that had begun to differentiate into alveoli and lobes, and secretion began to be present (Fig. 5B).
190
HERRENKOIIL AND CAMPBELL
Mann-Whitney U tests on mean S-A and L-A ratings revealed that pregnant collared stimulated animals had significantly greater secretory activity and lobuloalveolar development than at least two of its control groups (for the pregnant collared stimulated rats, cls = 64.0 and 58.5, n, = 9, n2 = 9, p < .002: for pregnant collared rats, Us = 62.0 and 45.2; n, = 9, n2 = 9, p < .002). However, they did not have significantly greater mean S-A and L-A ratings than the third control group (for pregnant collared etherized rats, Us = 16.5 and 15.5; nl = 10, nz = 9; p > .05). As expected, mean ratings of S-A and L-A in virgin stimulated animals were significantly greater than those of nonstimulated virgin rats (Us = 19.5 and 20.4; nl = 9; n2 = 9; p < .05). DISCUSSION Pregnant rats prevented from self-licking that received mechanical stimulation of the ventral body surface were observed to have mammary glands almost as well-developed as those of normally pregnant untreated rats. As expected on the basis of past results (Roth and Rosenblatt, 1966, 1968) both kinds of females had significantly greater mammary development than females similarly prevented from self-licking by collars around the neck (Birch, 1956). Moreover, mechanically-stimulated females had significantly greater mammary development than control females that were also prevented from self-licking, but that were either only restrained on the machine or placed under light anesthesia. Thus, by all the measures we employed, whether subjective or objective, mechanical stimulation of the ventral surface of pregnant rats prevented from self-licking was found to increase development of the mammary glands. The only minor and unexpected result was that ratings of glandular condition did not differ significantly between the primary experimental group and the etherized control group, due, most likely, to the larger individual differences in the latter. That stress of mechanical stimulation was the cause of the increase in mammary development appears unlikely for the following reasons. First, we found that neither ether nor restraint induced development of the mammary glands (Figs. 2, 3). Under a number of conditions, both these factors have been found to be stressors that enhance development of the mammary glands (Meites, Nicoll and Talwalker, 1963; Grosvenor, Krulick and McCann, 1968). Second, there were no signs that pregnancy was disturbed. If stress was active we would have expected to find that fetuses were either aborted or absorbed, that maternal adrenal gland weights were high, and that surviving young were underweight (Selye, 1950; Cronroos, Kappila and Soiva, 1963; Weir and Defries, 1963). None of these signs were apparent (Table 1). Thus the most likely explanation is that mechanical stimulation itself, and not secondary effects associated with it, was the cause of the increased development of the mammary glands.
FIGURE
FIGURE
4C
4D
MECHANICAL STIMULATION OF RAT MAMMARY GLANDS
193
That self-licking is an important stimulus for development of the mammary glands during gestation was first demonstrated by Roth and Rosenblatt (1966, 1968) when they prevented pregnant rats from self-licking by collars around the neck and found that mammary development was reduced. In an analysis of biochemical changes in the mammary glands, McMurtry and Anderson (1971) reported that the glands of pregnant rats prevented from self-licking by collars around the neck contained amounts of DNA and RNA that were significantly smaller than those in normally pregnant rats. Moreover, in an extensive study of prevention of self-licking employing a variety of techniques, Whitworth (1972) found that mammary glands were also underdeveloped in pregnant rats prevented from self-licking by selective thelectomy . Again, by all the measures we employed, virgin rats receiving stimulation had significantly greater mammary development than normal virgin rats.
194
HERRENKOHLANDCAMPBELL
MEClIANlCAL STIMULATION OF RAT MAMMARY GLANDS
195
Instead of containing only the atrophic duct system characteristic of virgin rats, stimulated virgins had mammary glands resembling those of the first trimester of gestation (see review by Cowie and Folley, 1961). Although no virgins in this report received restraint without mechanical stimulation, restraint as a factor in mammary growth can be ruled out on the basis that stress effects were not apparent in pregnant rats. Thus, it was shown that mechanical stimulation can induce mammary development in virgin rats, in the absence of gestational states. That mechanical stimulation can induce mammary development in virgin rats provides scientific support for a number of puzzling anecdotal reports. Zaks (1962), for example, claimed that massage of the teats in virgin heifers and goats caused mammary growth and secretion and that when applied to parturient heifers, it facilitated greater milk yield. Moreover, reports through the years have suggested that poultices applied to the teats, as well as rubbing, cause nipple development in “virgin” animals in a variety of mammals, including humans. It is possible, therefore, that virgin girls, animals, and even men, can secrete milk under mechanical means (Seifer, 1921; Selye and McKeown, 1934; Folley and Malpress, 1948; Zaks, 1962; Knott, 1970). Given the observation that mechanical stimulation increased mammary development in both virgin and pregnant rats, there are several questions remaining. One, what hormones are released by sensory stimulation which, in turn, cause growth of the mammary glands? Gonadotrophin secretion from the anterior pituitary gland is the most likely cause, either directly, through prolactin (which causes secretion of milk) and/or indirectly, through stimulation of ovarian hormones which, in turn, produce development of the mammary glands (see review by Cowie and Folley, 1961). One ovarian hormone, progesterone, has now been shown to be the response in a neuroendocrine reflex involving copulatory stimulation of the vaginocervical region in female rats (Dilley and Adler, 1968; Adler, 1969). Although prolactin seems the most likely choice, (because of its primary action on mammary gland cells), measurement of prolactin in the pituitary gland have not as yet revealed systematic changes as a function of self-licking (Grosvenor and Mena, 1969). Serum assays of prolactin involving more refiied techniques need to be done. An equally interesting question is the relationship between self-licking during gestation and subsequent maternal behavior. In 1956, Birch devoted only a few paragraphs in a much-cited theoretical paper to a description of an experiment he performed. He raised female rats with collars from weaning and found that at birth they ate 9% of their young, and that, failing to assume an appropriate nursing posture over the surviving young, the mothers allowed the young to die. Christopherson and Wagman (1965) have repeated the study in part, but instead of infant mortality, they found a less dramatic result: prevention of self-licking led only to a significant reduction in pup
196
HERRENKOIIL
ASD CAMPBELL
weight. These observations, in conjunction with the present report, suggest that one way prevention of self-licking during gestation can alter subsequent development of the young is by means of a lactational dysfunction associated with underdeveloped mammary glands.
ACKNOWLEDGMENTS We gnatefully acknowledge support for this research from a Biomedical Sciences Support Grant to Temple University and by National Institute of Child Health and Human Development Research Grant No. HD-04715 awarded to Lorraine Roth tlerrenkohl as principal investigator.
REFERENCES Adler, N. T. (1969). Effects of the male’s copulatory behavior on successful pregnancy of the female rat. J. Comp. Physiol. Psychol. 69, 613622. Benson, G. K., and Folley, S. J. (1957). The effect of oxytocin on mammary gland involution in the rat. J. Endocrinol. 16, 189-201. Birch, H. G. (1956). Sources of order in the maternal behavior of animals. Amer. J. Orthopsychiat.
26, 270-284.
Christophcrson, L:.. R., and Wagman, W. (1965). Maternal behavior in the albino rat as a function of self-licking deprivation. J. Camp. Physiol. qYscho2. 26, 270-294. Cowie, A. T., Folley, S. J. (1961). The mammary gland and lactation. In W. C. Young (Ed.), Sex and Internal Secretions, 3rd ed. Williams and Wilkins, Baltimore, MD. Cross, B. A. (1967). Neural control of oxytocin secretion. In L. Martini and W. F. Ganong (Ms.), Neuroendocrinology. Academic Press, New York. Dilley, W. G., and Adler, N. T. (I 968). Postcopulatory mammary gland secretion in rats. Sot. Exp. Biol. Med. 129, 964-966.
Folley, S. J., and Malpress, P. H. (1948). Hormonal control of mammary growth. In G. Pincus and K. V. Thimann (Fds.), 7he Hormones. Academic Press, New York. Gronroos, M., Kauppila, O., and Soiva, K. (1961). Changes in the vaginal smears of rats during normal and disturbed pregnancies. Actu Endocrinol. 37, 309-314. Grosvenor, C. E. (1965). lvidence that exteroceptice stimuli can release prolactin from the pituitary gland of the lactating rat. Endocrinology 76, 340-342. Grosvenor, C. E., Krulich, L. and McC~M, S. (1968). Depletion of pituitary concentration of growth hormone as a result of suckling in the lactating rat. Endocrinology 82, 617619. Grosvenor, C. E., and Mena, F. (1969). Failure of self-licking of nipples to alter pituitary prolactin concentration in lactating rats Horm. Behov. I, 85-91. Knott, J. C. (1907). Abnormal lactation in the virgin; in the old woman; in the male; in the newborn of either sex (“witches milk”). Amer. Med. 2, 373. Lehrman, D. S. (1961). Hormonal regulation of parental behavior in birds and infrahuman mammals. In W. C. Young (Ed.). Sex and Internal Secretions, 3rd ed. Williams and Wilkins, Baltimore, MD. McMurtry, J. P. and Anderson, R. R., (1971). Prevention of self-licking on mammary gland development in pregnant rats. Proc. Sot. Exp. Bio. Med. 137, 354-356. Meites, J. NicoII, C. S., and TaIwaIker, P. K. (1963). The central nervous system and the secretion and release of prolactin. In A. V. Nalbandov (Ed.), Advances in Neuroendocrinolofl, University of Illinois Press, Urbana, IL.
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OF RAT MAMMARY GLANDS
197
Oshima, %l., and Goto, T. (1955). A study of involution of mammary gland after weaning in the albino raL. Bull. :l;atr~ host. Agric. Sci., Tokyo (Ser. G), 11, 81-87. Rheingold, 11. (Ed.). (1963). Maferrlal Behavior in Mammals, John Wiley and Sons, New York. Kosenblatt, J. S., and Lehrmdn, D. S. (1963). Maternal behavior of the laboratory rat. In II. Rheingold (Ed.), Marernal Behavior in Mammals. John Wiley and Sons, New; York. Roth, 1.. L., and Rosenhla~t, J. S. (1966). Mammar) r glands of pregnant rats: Development stimulated by licking. Science 151, 1403-1404. Roth, L. L., and Rosenblatt, J. S. (1967). Changes in self-licking during pregnancy in the rat. J. Cbrnp. PhysioL Psychol. 63, 397-400. Roth, L. I.., and Rosenhlatt, 1. S. (1968). Self-licking and mammary development during pregnancy in the rat. .I. b,Wocrinol. 42, 363-378. Seifert, M. J. (1920). Abnormal lactation. J. Anzer. !Med. Assoc. 74, 1634. Selye, Hans (1950). Stress. Acta Incorporated, biontreal, Canada. Selye, II., and YcKeown, T. (1934). Production of pseudo-pregnancy by mechanical stimulation of the nipples. Froc. Sot. Exp. Biol. 31, 638-687. Squartini, t;. (1957). A new method for the study of the mammary gland of the mouse. Cancer 10, 179-182. Weir, M. W., and De I:rics, J. C. (1963). Blocking of pregnancy in mice as a function of stress. Ps~~clzol.Repts. 13, 365-366. Whitworth, Neil (1972). Relationships between patterns of grooming, endocrine function and mammary gland development in the pregnant rat. Doctoral dissertation, submitted to Rutgers University, New Brunswick, NJ. Zaks, M. G. (1962). ‘Uze Motor Apparatus of the Mammary Gland. Translated by D. G. Fry., A. T. Cowie (Ed.). Oliver and Hoyd, London.