Influence of Hormones on Synthesis and Secretion of Milk Proteins by Mammary Tissue from Male and Female Cattle of Beef and Dairy Breeds

INFLUENCE OF HORMONES ON SYNTHESIS AND SECRETION OF MILK PROTEINS BY MAMMARY TISSUE FROM MALE AND FEMALE CATTLE OF BEEF AND DAIRY BREEDS

Thomas Bernard McFadden

Thesis submitted to the Faculty of the

Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Dairy Science

APPROVED:

R.M. Akers, Chairman

W.E. Beal F.C. Gwazdauskas

J.M. White, Department Head

March, 1985 Blacksburg, Virginia INFLUENCE OF HORMONES ON SYNTHESIS AND SECRETION OF MILK PROTEINS BY MAMMARY TISSUE FROM MALE AND FEMALE CATTLE OF BEEF AND DAIRY BREEDS

Thomas B. McFadden

(ABSTRACT)

The ability of mammary tissue from prepubertal bulls

and heifers of beef and dairy breeds to respond to hormonal

stimuli through synthesis and secretion of milk proteins was

studied. Experimental animals were six to eight month old

Angus and Holstein cattle. All subjects were injected with

estradiol and progesterone for seven days and slaughtered on

day 15. Mammary tissue was explanted and cultured for 96 h

in basal medium (B) which contained hormones necessary for

maintenance, or stimulatory medium (P), further supplemented

with prolactin. Selected cultures were incubated for 24 h

in B or P medium containing 3 H-amino acids. Concentrations

of non-labeled alpha-lactalbumin (Alac), 3 H-Alac, and

3 H-total protein (TP) were determined in media and in ex-

plant homogenates.

Among cultures of bull mammary tissue, Angus explants

secreted greater overall quantities of 3 H-TP and 3 H-Alac than Holstein explants (p<.05). Secretion of Alac was also greater in Angus cultures at two of eight treatment periods

(p<.01). Concentrations of all three protein fractions were likewise enhanced in homogenates of Angus explants for at least three of four treatment periods (p~.05). Presence of prolactin in medium stimulated secretion of Alac (p<.005), and accumulation of all three fractions in explants (p<.10).

Holstein heifer explants secreted more Alac at three of eight treatment periods than Angus explants (p<.0005). Ove- rall secretion of 3 H-TP and 3 H-Alac also was elevated in

Holstein over Angus females (p<.10), as were concentrations of all three fractions in homogenates (p~.01). Presence of prolactin had no direct effect on any protein parameters in female tissue.

I conclude that mammary tissue of immature bulls and heifers can be hormonally induced to express it's genetic merit for milk production (based on breed differences), through synthesis and secretion of milk proteins. Prolactin

stimulated protein production in bulls but not in heifers.

These findings indicate that similar methods of stimulating mammary tissue to produce milk proteins may be adaptable for commercial evaluation of genetic potential for milk production, especially in young bulls. ACKNOWLEDGEMENTS

I would like to express my sincere appreciation to the following people, who were instrumental in helping me pre- pare this thesis:

Dr. R.M. Akers - I am convinced that I couldn't find a better major professor anywhere, which explains why I'm staying on for a PhD. Need I say more?

Dr. W.E. Beal - For many and varied instances of assis- tance and encouragement, but particularly for his enthusiasm toward, and philosophies of teaching.

Dr. F.C. Gwazdauskas - For much valuable classroom in- struction as well as encouragement and advice throughout my graduate studies.

Dr. J.M. White - For exceptional enthusiasm and support toward myself and the entire department, not to mention some good jokes.

Many thanks and a few apologies are due my wife, Holli, who more than anyone bore the brunt of my own special brand of thesis blues. Her constant love and encouragement prac- tically earned her a co-authorship.

Finally I thank God for giving me the health and strength to complete this task.

iii TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGEMENTS iii

Chapter

I. INTRODUCTION 1

I I. REVIEW OF LITERATURE 3

Induced Lactation and Mammary Growth 3 Hormonal Control of Lactogenesis: Laboratory Species ...... 18 Hormonal Control of Specific Protein Synthesis, In Vitro ...... 24 Hormonal Control of Lactogenesis: Ruminants 28 Lactation in Male Mammals 37

III. MATERIALS AND METHODS 42

Animals . 42 Hormone Pretreatment of Subjects 43 Organ Culture of Mammary Explants 43 Incubation Medium .... 44 Incubation Protocol 46 3 H-Thymidine Incorporation 48 3 H-protein Quantification 49 Protein Radioimmunoassays (RIAs) . 52 Statistical Methods 52

IV. RESULTS . . . 53

Experiment I. Males 53 Media Alpha-lactalbumin ...... 53 3 H-Total Protein and Alpha-lactalbumin in Media ...... 54 3 H-Total Protein and Alpha-lactalbumin in Homogenates ...... 56 Non-radiolabeled Alpha-lactalbumin Concentrations in Media and Explant Homogenates ...... 60 Synthesis versus Secretion of Proteins by Cultured Mammary Explants from Angµs and Holstein Bulls 64 Correlations 71

iv 3 H-Thymidine Incorporation into Mammary Explant DNA...... 71 Serum Prolactin and Alpha-lactalbumin 73 Experiment II. Females ...... 73 Media Alpha-lactalbumin...... 73 3 H-Total Protein and Alpha-lactalbumin in Media ...... 75 3 H-Total Protein and Alphalactalbumin in Explant Homogenates ...... 79 Non-radiolabeled Alpha-lactalbumin in Media and Homogenates ...... 83 Synthesis versus Secretion of Proteins by Cultured Mammary Explants from Angus and Holstein Heifers . . . . 86 Correlations ...... 90 3 H-Thymidine Incorporation into Mammary Explant DNA...... 92 Serum Prolactin and Alpha-lactalbumin 92 v. DISCUSSION 96 Experiment I. Males 96 General 96 Breed Differences . 97 Effect of Treatment and Incubation Time on Productive Indices . . . . . 102 Synthesis versus Secretion of Milk Proteins 104 Other Considerations 106 Experiment II. Females 111 General ...... 111 Breed Differences. 112 Effect of Treatment on Protein Synthesis and Secretion ...... 113 Synthesis versus Secretion of Milk Proteins 116 Other Considerations 118

VI. SUMMARY AND CONCLUSIONS .. 121

LITERATURE CITED 125 VII. APPENDIX A 134

V LIST OE' TABLES

Table

1. Synthesis and secretion of milk proteins by cultured mammary explants from Angus and Holstein bulls .. 68

2. Incorporation of 3 H-thymidine into DNA of mammary explants from Angus and Holstein bulls. . . 73

3. Concentrations of prolactin in serum of Angus and Holstein bulls during steroid pretreatment. 75

4. Synthesis and secretion of milk proteins by cultured mammary explants from Angus and Holstein heifers. 90

5. Incorporation of 3 H-thymidine into DNA of mammary explants from Angus and Holstein heifers. . . 94

6. Concentrations of prolactin in serum of Angus and Holstein heifers during steroid pretreatment ... 95

vi LIST OE' FIGURES

Figure 1. Secretion of Alac into medium by mammary explants from Angus and Holstein bulls...... 55

2. Secretion of 3 H-TP into media by Angus and Holstein bull explants...... 57

3. Secretion of 3 H-Alac into media by Angus and Holstein bull explants...... 58

4. Concentrations of 3 H-TP in homogenates of Angus and Holstein bull explants ...... 60

5. Concentrations of 3 H-Alac in homogenates of Angus and Holstein bull explants ...... 61 6. Secretion of non-radiolabeled Alac into media by bull explants exposed to 3 H-amino acids during culture...... 64 7. Concentrations of non-radiolabeled Alac in homogenates of bull explants exposed to 3 H-amino acids during culture ...... 65 8. Secretion of Alac into medium by mammary explants from Angus and Holstein heifers...... 76

9. Secretion of 3 H-TP into media by Angus and Holstein heifer explants...... 78

10. Secretion of 3 H-Alac into media by Angus and Holstein heifer explants...... 79

11. Concentrations of 3 H-TP in homogenates of Angus and Holstein heifer explants ...... 82

12. Concentrations of 3 H-Alac in homogenates of Angus and Holstein heifer explants ...... 84 13. Secretion of non-radiolabeled Alac into media by heifer explants exposed to 3 H-amino acids during culture...... 87

vii 14. Concentrations of non-radiolabeled Alac in homogenates of heifer explants exposed to 3 H- amino acids during culture...... 88

viii Chapter I

INTRODUCTION

Mammary growth and milk production are highly-integrated, hormonally- regulated developmental processes, which ultimately allow the mammalian mother to provide for the nour- ishment of her offspring. In the dairy cow, a lactating female provides milk not only for her progeny, but also for a host of "symbiotic" humans as well, thus forming the basis of the dairy industry. It is this outside demand for milk which has lead to the extreme specialization of dairy breeds, and which exerts a profound demand for ever-increas- ing efficiency of milk production. The critical role of hormones and genetic merit of individual animals in determining milk yield has stimulated much research activity; hormonal induction of lactation and sire-selection programs being primary examples of each.

Because hormones play an essential role in eliciting expression of genes involved in production of milk, endocrinology and genetics may be studied as integrated elements directed toward a common goal, milk production. This concept formed the basis for the present study. Expression of differential in genetic potential for milk production, measured by synthesis and secretion of milk proteins, was stu-

1 2

died through hormonal manipulation of mammary tissue from high- (dairy) and low- (beef) yielding breeds, in vivo and in vitro. Thus the primary objective of this thesis was to develop a "bioassay" for genetic potential to produce milk.

Such a system might find applications in the sire selection industry, and also yields useful information on hormonal control of genes involved in milk production. Maximal selection for, and expression of these genes are the major goals of research related to the dairy industry. Chapter II

REVIEW OF LITERATURE

INDUCED LACTATION AND MAMMARY GROWTH

The influence of the reproductive tract on the development of the mammary gland has long been apparent. Increased mammary growth at puberty followed by more extensive development during pregnancy has been a clear indication of the relationship between reproductive status and mammary development. Early study of this relationship suggested that estrogen and progesterone were intimately associated with growth of the mammary gland. The effects of these hormones have thus become a focal point for research on both normal and artificial development of the mammary gland.

Much of the early research on mammary growth involved characterization of steroid-induced development in various species. In castrated male and female rats injection of

"estrus-producing hormone" plus extract of corpora lutea produced ductular and lobular proliferation. Mammary development was less than that present in glands from females in advanced pregnancy, but greater than that of animals receiving estrus- producing hormone alone (Turner and Schultze,

1931). Turner and DeMoss (1934) reported that administration of theelin (an estrogenic compound) caused extensive

3 4

ductular development of the mammary gland in castrated female and intact male cats. Subsequent treatment with corporin (an extract of corpora lutea containing progestin) resulted in stimulation of lobule-alveolar development equiva- lent to that of mid-pregnant females. Males treated with galactin (a crude pituitary prolactin preparation) exhibited initiation of glandular development and milk secretion comparable to that observed in parturient females. Similar results were obtained in rabbits (Turner and Frank, 1932), and in guinea pigs and mice (Turner and Gomez, 1934a,b). Injec- tion of estrogen into ovariectomized female and intact male dogs stimulated only slight ductular growth, however this result was attributed to inadequate dose rather than lack of hormonal effect (Turner and Gomez, 1934c). Asdell et al.

(1936), injected estrone and progestins into castrated rabbits, some of which had been hypophysectomized. These workers observed much greater mammary development in treated animals, regardless of presence of hypophyses, than in a castrated control, and concluded that ovarian hormones are capable of stimulating mammary growth independent df pituitary influence. In an attempt to better understand the ability of injected steroids to stimulate mammary development, Meites and Turner (1948), studied the effect of progesterone or estrogen on blood and pituitary lactogen con- 5

tent. Injection of progesterone in near-physiological doses did not increase pituitary lactogen content in guinea pigs or rats, however supraphysiological doses induced increases of 25-100%. Injection of estrone into male rabbits caused

simultaneous increases in blood and pituitary lactogen content, demonstrating that increased secretion accompanies in-

creased pituitary content of lactogen. These studies clear-

ly indicate that steroids are able to stimulate mammary growth directly, and further suggest that onset of lactation per se might be induced through synergism of pituitary lac- togens with ovarian steroids.

The above experiments demonstrated the stimulatory ef-

fects of estrogen and progesterone treatment on growth and development of the mammary gland, thus laying the groundwork

for further studies concerned with applying similar treatments to dairy animals. The objectives of this research were to increase mammary development and subsequent milk production as well as to induce lactation in non-milking an-

imals and further to elucidate the action of steroids in dairy animals. Hammond and Day (1944) implanted 140 cows

and heifers which had failed to conceive, with stilboestrol

or hexoestrol tablets. Stimulation of mammary growth was

observed in heifers, and lactation was initiated in a major-

ity of cows and heifers although yields were highly variable. Lactating cows ceased lactating following treatment. 6

In a similar study, Folley and Malpress (1944a) induced lactation in nulliparous heifers and dry cows by subcutaneous implantation of diethyl stilboestrol or hexoestrol tablets. Although individual variation was great, some animals yielded milk in quantities comparable to that of normal lactation. Variability in response was attributed to variable rates of tablet absorption and heterogeneity in the po- pulation of experimental animals. In addition these authors suggested a requirement for progesterone as well as estrogen for full alveolar development. This experiment also described deleterious side effects of estrogen treatment including nymphomania and weakening of the pelvic girdle which led to pelvic fracture in 20% of the subjects. A subsequent attempt by these authors (Folley and Malpress, 1944b) to orally administer estrogens was largely unsuccessful. Only

59% of the subjects yielded greater than 1 lb per milking and yields were generally inferior to those obtained following parturient lactation, although results were variable.

The effective dose needed orally was much greater than that required via injection thus oral administration of estrogens was deemed economically unfeasible.

Histological evaluation of udder development in heifers in response to cornbir.ations of diethylstilboestrol, progesterone, and pituitary extract was performed by Sykes and 7

Wrenn (1951). These workers noted ductular and alveolar development which was abnormal following treatment with diethylstilboestrol alone, but improved with the addition of pituitary extract and appeared nearly normal when all three hormones were applied. Interestingly, they observed that amount of secretion obtained was inversely proportional to the histological normality of the tissue. In a similar study, Cowie et al. (1952) administered varying doses of estrogen alone, or combined with progesterone to observe milk yield and histological development of castrated female dairy goats. Estrogen stimulated mammary growth causing visible swelling of the udder, and induced milk secretion, while estrogen and progesterone combined resulted in less swelling and initially lower milk yield. Udder size and milk yields of animals receiving the combination treatment increased to levels equal to or greater than those of animals treated with estrogen, however yields from both treatment groups were lower than yields expected during lactation following parturition. Histological assessment of glands from estrogen-treated donors indicated a deficiency in total area of secretory epithelium, numerous small folds in alveolar and ductular tissue, and abnormally large alveoli. These abnor- malities were rarely observed in tissue from estrogen plus progesterone treated goats. The atypical histological de- 8

velopment and rapid initiation of milk secretion associated with estrogen treatment is in contrast to the normal growth and slower onset of milk production observed when progesterone is also injected, similar results have been previously reported in heifers (Sykes and Wrenn, 1951). It is clear then that progesterone moderates the rapid growth initiated by estrogen treatment resulting in a more histologically normal gland. In addition, Cowie et al. (1952) postulated that in goats, a dose level of estrogen that is both mammo- genic and lactogenic when administered alone, can be deprived of its lactogenic effect when in the presence of progesterone. This may account for the inverse relationship between milk yield and histological normality observed by

Sykes and Wrenn (1951), since their measurements of milk production were made during the treatment period when lactogenesis may have been blocked in animals receiving progesterone.

Turner et al. (1956) primed eight dairy heifers with injections of estradiol benzoate plus progesterone for 180 days to stimulate lobule- alveolar development, then administered estrogen alone for 14 or more days to induce lactogenesis. These animals were successfully induced to lactate, producing 66-137% of that produced by their maternal and paternal half-sisters. Maximum milk yields attained 9

ranged from 5.5 to 15.1 kg per day. Thus injection of estrogen alone to induce lactogenesis in udders developed with estrogen plus progesterone appeared to better mimic parturient lactation than did earlier induction techniques. Wil- liams and Turner (1961) investigated the effects of dosage and duration of estrogen-progesterone priming on milk yields of 20 heifers induced into lactation with estrogen alone for

14 days. Average maximum daily milk yields from 14 heifers injected with 200 ug estradiol benzoate plus 200 mg progesterone per day for 120 or 180 days were 9.6 and 10.8 kg per day respectively, compared to 10.3 kg per day from animals on previous experiments (Turner et al., 1956) that received

100 ug estradiol benzoate plus 100 mg progesterone per day for 180 days. Thus neither dosage administered nor duration of the priming period significantly affected subsequent milk yield. The six remaining heifers were primed with the higher dosage for 60 days, of this group the three highest pro- ducers averaged 9.7 kg per day whereas the three lowest pro- ducers averaged 4.6 kg per day. The authors suggested that the more productive animals may have undergone some lobuloalveolar development during previous estrous cycles and that steroid priming was effective in continuing this growth.

Initial development in the less productive heifers may have been retarded, thus after the priming period they remained 10

underdeveloped for milk production relative to their group- mates. Neller and Reineke (1958) found that sexually mature

dairy goats were more responsive to induction of lactation with progesterone and stilboestrol than 8 month old goats.

In addition, they reported that goats receiving the combina-

tion of hormones displayed a more rapid onset of lactation

than those treated with stilboestrol alone, in contrast with

the reports of Cowie et al. (1952). They further suggested

that variation in response among individual animals might

preclude the assignment of any particular dose or ratio of hormones to be universally accepted as the optimum for in-

ducing lactation. By contrast, Sud et al.(1968) found that

absolute dose of progesterone and estrogen was more critical

in stimulating mammary development in ovariectomized heifers

than was the ratio of hormones administered. The two most

responsive groups however, received the same ratio of estro-

gen:progesterone at differing doses, suggesting the exis-

tence of an optimal ratio within a certain range of doses.

Although the treatment employed in their study did not in-

duce lactation, histological growth indices from optimally

treated heifers were similar to those from pregnant control

heifers.

The first use of adrenal cortical steroids to induce lac-

tation was reported by Tucker and Meites (1965). These 11

workers successfully initiated lactation in early, mid-, and late pregnancy dairy heifers, utilizing injections of

9-fluoroprednisolone acetate, a synthetic glucocorticoid, followed by twice-daily milking. This research demonstrated the importance of adrenal cortical steroids in initiating lactation, and further showed that even relatively undevel- oped (three and one-half months of pregnancy) glands were capable of milk secretion.

The results of these studies on dairy animals added a great deal to understanding of the growth and development of the mammary gland. Key contributions included description of the role of progesterone, discovery of glucocorticoid effects, and theory on growth during estrous cycles. However, attempts to optimize an induced lactation technique through manipulation of dose, hormone ratio, and duration of treatment did not result in a reliable procedure for initiation of lactation. Marked variation was evident, extensive treatment periods required, and indeed even the most suc- cessful artificial lactations were generally inferior to yields expected, had the animals undergone parturient lactation. For these reasons artificial induction of lactation remained for most purposes, imprac~icable.

A major advance occurred when Smith and Schanbacher

(1973) initiated lactation in seven of ten nonpregnant, dry 12

cows by injecting .1 mg estradiol plus .25 mg progesterone per kg bodyweight, daily for 7 days. This protocol had been previously employed to induce irnrnunoglobulin transport in

the mammary glands of dry cows (Smith et al., 1971). Furth-

er studies demonstrated that nulligravid heifers could be

successfully induced to lactate following similar treatment.

In addition, neither total amount nor ratio of estradiol and

progesterone proved to be a critical determinant of the suc-

cess of initiating lactation (Smith and Schanbacher, 1974).

Clearly this method of inducing lactation was more practical

than early methods requiring 60-180 days of treatment (Tur-

ner et al., 1956).

Researchers attempting to characterize the changes in en-

docrine patterns associated with the abbreviated induction

scheme of Smith and Schanbacher (1973), observed a relative-

ly rapid turnover of injected hormones. Ninety percent of

injected progesterone was excreted within 17 days, while 88%

of estradiol and 94% of injected progesterone were excreted

by 28 days post-injection. Major routes of excretion were through feces and urine with very little recovery from milk

(Willett et al., 1976). Hormonal profiles from cows suc-

cessfully induced into lactation were characterized by a ra-

pid decline in plasma concentrations of estrogen and pro-

gesterone after cessation of injections, and low 13

concentrations thereafter. Additionally, positive associations between plasma prolactin concentrations and milk yields were recorded (Erb et al., 1976; Mollett et al.,

1976). Erb et al. (1976) found hormonal profiles following treatment with estradiol plus progesterone for 7 or 12 days to be similar to profiles from pregnant cows 3 to 7 days prepartum, but deficient in absolute concentrations of plasma estrogen and prolactin compared with parturient cows.

These results suggest that tremendous acceleration in growth and differentiation of the mammary gland occurs in response to the relatively short-lived hormonal stimulus of steroid treatment. This stimulus apparently mimics the hormonal milieu present during gestation and the pre-partum period, and yet is able to cause growth and initiate lactation in a comparatively short time. The active hormones for growth pro- motion are thought to be estrogen and progesterone, while prolactin is strongly implicated as well, and may be particularly important in differentiation of the secretory cells and actually initiating lactation.

Armed with a clearer understanding of the endocrine relationships during induced lactation, several workers attempted to improve results by modifying the treatment administered. Tucker and Meites (1965) report on the importance of glucocorticoids in initiation of lactation in pregnant heif- 14

ers indicated that addition of glucocorticoids might improve responsiveness. Erb et al. (1976) also hypothesized that estrogen-progesterone treatment should begin 3-8 days after estrus. Accordingly, Howe et al. (1975) modified the induction scheme of Smith and Schanbacher (1973), by initiating treatment in non-pregnant, non-lactating, multiparous cows 7 days post-estrus and injecting dexamethasone, a synthetic glucocorticoid, on days 12, 13, and 14, after estrogen-progesterone treatment. Histological evaluation of mammary tissue biopsied on day 23 after initiation of treatment indicated that tissue development was similar to that of

2-week, but less than that of 2-day prepartum tissue. Milk yields from animals not biopsied ranged from 33-74% of previous lactations. Thus addition of glucocorticoid to the treatment regime did not appreciably improve yield or decrease variability as compared to the previous studies of

Turner et al. (1956) and Erb et al. (1976). Collier et al.

(1977) extended this approach by injecting reserpine, an alkaloid, tranquilizing agent shown to elevate prolactin concentrations in serum, in addition to the estrogen-progesterone and dexamethasone combination used previously (Howe et al., 1975). Cows thus induced to lactate had elevated prolactin concentrations during treatment and subsequently attained higher peak yields and greater milk production over 15

100 days than controls not receiving reserpine. These results clearly suggested that increased prolactin concentrations might dramatically improve the success of steroid therapy for inducing lactation. Further study however, has been unable to detect a stimulatory effect of reserpine injections (Skarda et al., 1983). In spite of evidence to the contrary, the importance of prolactin in eliciting lactogenesis in steroid therapy-induced lactation has been defi- nitively shown through studies employing the prolactin anta- gonist CB-154 (bromocriptine, a synthetic ergot alkaloid).

Schams (1976) reported that lactation was successfully induced in heifers treated with estradiol, progesterone and dexamethasone, however twins of the steroid treated animals, which received CB-154 injections in addition to the steroids, had reduced prolactin concentrations in serum and failed to lactate following treatment. Similar findings have been reported in goats by Hart and Morant (1980).

These investigators found that goats treated with CB-154 in addition to estradiol benzoate and progesterone had reduced concentrations of prolactin in serum and inhibited milk secretion relative to control animals treated with steroids alone. The above findings are indicative of a specific requirement for prolactin in lactogenesis in cattle and goats. 16

After some 50 years of research, the lack of success in defining a reliable, practical procedure for induction of lactation is regrettable, especially considering the potential for applications in the dairy industry. This research however, has been of tremendous value in theoretical understanding of mammary development. In addition, these studies find applications in experiments on periparturient changes involved in the onset of lactation. The composition of milk obtained from cows and heifers induced to lactate has been described as normal in terms of fat, protein, and total solids (Skarda et al., 1983), as well as in content and composition of whey proteins (Becka et al., 1983). Folley and

Malpress (1944c) found that the composition of milk from estrogen-induced heifers changed from colostral to normal milk as production increased, similar to the changes observed in parturient lactation. These findings support the idea that this milk is the product of a normally functioning mammary gland, and is consequently a suitable model for study of

"normal" development. Collier et al. (1976) studied the relationship between biosynthetic capacity of tissue obtained by biopsy of cows induced to lactate, and their subsequent milk production.

Ability to synthesize lactose and fatty acids was determined by incubation of mammary tissue slices from each animal. 17

Observed differences between animals in these biosynthetic measurements corresponded to differences in subsequent milk production. Additionally, Croom et al. (1976) investigated histological changes occurring in the same cows (Collier et al., 1976). Histological evaluation showed an association between ultrastructural development of tissue from a particular animal and the success of its ensuing lactation. Tis- sue from cows responding favorably to hormonal stimulation formed dense areas of alveolar tissue and underwent cellular changes such that cytological organization came to resemble that of normally lactating bovine mammary tissue. These essential changes did not occur in less responsive animals.

Nickerson et al. (1978) cultured tissue from heifers treated with estradiol and progesterone according to the technique of Smith and Schanbacher (1973) to investigate the ability of various hormones and plasma to stimulate histological development in vitro. Incubation of tissue with insulin, cortisol, estrogen and progesterone preserved initial morphology, while lactogenesis was induced in tissue cultured in the presence of insulin, cortisol and prolactin, and further enhanced by the addition of plasma to this combination.

Ichinose and Nandi (1964) were able to induce lobulo-alveolar growth comparable to late pregnancy with mammotrophic 18

hormones in vitro, in mammary tissue from immature mice treated in vivo with estrogen, progesterone, ovine mammotro- phin and bovine somatotrophin, but not in mice not receiving in vivo treatment. These studies are cited as examples of the usefulness of mammary tissue obtained from steroid- treated animals in studies of lactogenesis.

Clearly the study of induced lactation and steroid effects on mammary growth have been most profitable to the understanding of mammary gland development. In addition, techniques of induced lactation are readily adapted to basic studies of mammary growth and differentiation, since the tissue thus obtained, in combination with in vitro techniques, is perhaps the best available artificial model for study of normal lactogenesis.

HORMONAL CONTROL OF LACTOGENESIS: LABORATORY SPECIES

Lactation can be defined as the copious secretion of milk. The developmental processes necessary for full lactation may be divided into three stages: mammogenesis or mammary gland growth, lactogenesis, the initiation of milk secretion, and galactopoiesis, the maintenance of secretion

(Schams, 1976). The first two of these stages are appropriate for review within the context of the present study.

Much of the current understanding of mammogenesis and lacto- 19

genesis is based on studies of induced lactation, endocrine gland ablation followed by replacement therapy, and in vitro culture experiments. Research on induced lactation, particularly in dairy animals has been of importance in defining the in vivo role of ovarian steroids, glucocorticoids and prolactin in growth and differentiation of the mammary gland, as reviewed in the previous section. In vitro studies have been especially effective in gaining insight on the onset of lactation in mammals.

An important point to consider in evaluating in vitro experiments is whether results thus g?ined are a valid repre- sentation of in vivo events. Forsyth (1971), states that results obtained from in vitro studies generally agree very well with in vivo studies performed on triply- operated

(adrenalectomized, hypophysectomized, ovariectomized) laboratory animals. In one such study, Lyons et al. (1958) were able to induce full lobuloalveolar differentiation in triply operated rats by administering prolactin, growth hormone, estrogen, progesterone and corticoid. Prolactin and corticoid therapy was the minimal requirement for lactogenesis, however no single hormone was capable of inducing full lobule-alveolar development. Similar results have been reported in triply-operated mice (Nandi, 1959). In addition, an experiment by Welsch et al. (1979) showed that a combina- 20

tion of prolactin, growth hormone and hydrocortisone increased alphalactalbumin content of bovine mammary tissue transplanted into athymic nude mice previously treated with estrogen, progesterone, growth hormone and prolactin. These studies have formed the basis for acceptance of in vitro experiments on hormonal effects on lactogenesis.

Initial experiments utilizing in vitro culture of mammary gland explants sought to define the hormonal combination needed to induce lactation, that is, the lactogenic complex.

Elias, (1959) first reported an absolute requirement for insulin to maintain structural integrity of the mouse mammary gland in vitro. Ichinose and Nandi, (1964) pretreated female mice with injections of estradiol, progesterone, ovine mammotropin and bovine somatomammotropin for 7 days, then cultured the mammary glands in vitro. Glands incubated 5 days in medium supplemented with insulin, mammotropin and

somatomammotropin displayed lobule-alveolar development comparable to that seen in late pregnancy, an additional 5 days of culture in "secretion-inducing hormones" (aldosterone or cortisol, mammotropin, somatomammotropin and insulin) re-

sulted in histological evidence of highly active secretion.

Barnawell, (1965) examined the in vitro histological response to hormones of mammary tissue from rat, hamster, guinea pig, rabbit and dog. All animals were postpubertal fe- 21

males pretreated with estrogen and progesterone to induce

lobule-alveolar development. Medium supplemented with insu-

lin enhanced survival of tissue from all species without

concurrently stimulating secretory activity. Further sup-

plementation with corticosteroid yielded better survival in

all species. Addition of ovine prolactin stimulated secre-

tion in all species but guinea pig; dog and rabbit were

especially responsive. The above experiments provided good histological evidence for the idea that maintenance of mammary cell integrity was dependent upon exposure to insulin

plus corticoid, and that the addition of prolactin specifi-

cally induced secretion. While results from the histologi-

cal evaluations used in these studies were strongly indica-

tive of changes associated with terminal differentiation,

direct evidence in terms of a biological endpoint was lack-

ing. Juergens et al. (1965) were among the earliest re-

searchers to evaluate lactogenic response based on biochemi-

cal measurements. These workers utilized incorporation of

radioactive amino acids into "caseinlike" phosphoproteins as

an index of protein synthesis in mammary tissue from mid-

pregnant mice cultured in medium containing insulin, hydro-

cortisone and prolactin. Protein synthetic rates after 48

hours in culture were increased several fold compared to in-

itial rates of synthesis. Medium supplemented with any of 22

these hormones alone or in pairs elicited little or no stimulation, however any combination including insulin was second in effectiveness to the triple combination, while combinations excluding insulin were no more effective than unsup- plemented medium. Voytovich and Topper (1967) followed up these experiments by demonstrating that tissue from untreated immature mice could be induced to synthesize the milk proteins casein and alphalactalbumin in the absence of lobule-alveolar structures, when exposed to insulin, hydrocortisone and prolactin in vitro. In confirmation of Ichinose and Nandi (1964) in vivo priming of donors with estrogen, progesterone and growth hormone was necessary to induce development of alveolar structures in the ensuing in vitro incubation. These results, as well as those of Vonderhaar and

Smith, (1982) demonstrated that structural development may be dissociated from biochemical differentiation in vitro.

Ceriani (1970a,b) incubated explants of mammary gland anla- gen from fetal rats and found that the hormonal combination of insulin, aldosterone, prolactin and progesterone was the most effective in promoting growth, as determined by histological assessment, and biochemical differentiation, as measured by synthesis of caseinlike material. Histological evaluation was in good agreement with biochemical indices of development. Thus even the embryonic rudimentary mammary 23

gland is capable of responding to hormonal stimulation in a manner analogous to that seen in mammary glands from adult animals. Turkington et al. (1968) demonstrated that prolactin stimulated synthesis of both the A (galactosyl transferase) and B (alphalactalbumin) proteins of the lactose synthetase enzymatic complex, following preincubation of midpregnant mouse mammary tissue with insulin and hydrocortisone. The concentrations of these proteins, as measured by specific enzymatic assay, were comparable to those observed in mammary tissue from periparturient mice. These studies unanimously support the view that insulin, corticoid and prolactin supplementation of medium supplies a lactogenic complex suitable to induce measurable differentiation in mammary tissue from several laboratory species. Cytological and biochemical differentiation in vitro generally develops concurrently, as in parturient lactogenesis in vivo. Howev- er, the findings of Voytovich and Topper, (1967), as well as those of Vonderhaar and Smith, (1982), serve as a reminder that all in vitro systems are artificial and warn that manipulation of such systems may yield results not strictly compatible with observations of in vivo development. 24

Hormonal Control of Specific Protein Synthesis, In Vitro

Hormonal control of lactogenesis has been extensively studied through measurement of protein synthetic response to lactogenic hormones in vitro. Perhaps the two most commonly measured proteins are casein and alphalactalbumin. With the emergence of sensitive assay procedures, specific hormonal influences on synthesis of these proteins individually, as well as in relation to each other, have been elucidated.

Turkington et al. (1968) noted that synthesis of the A and B proteins of the lactose synthetase complex proceeds asynch- ronously during gestation and into lactation. The A protein is synthesized more rapidly than B throughout gestation, however as lactation commences a dramatic rise in B-protein synthesis occurs. Perhaps A-protein synthesis represents a preparatory phase of development which is shifted to an initiating phase as B-protein synthesis accelerates, providing a large pool of lactose synthetase at the onset of copious milk secretion. Vonderhaar et al. (1973) found that in mammary gland explants from mature virgin mice cultured in the presence of insulin, hydrocortisone and prolactin, activity of the lactose synthetase A-protein and casein synthesis peaked 24 hours earlier than did activity of the B-protein, alphalactalbumin. Priming of donors with estradiol or prolactin however, caused protein syntheses to peak simultane- 25

ously, reflecting the synchrony observed in explants from pregnant mice. The results of these studies suggest that syntheses of specific milk proteins are controlled by complex hormonal interactions which may operate independently for different proteins. That differential effects of hormones on protein syntheses and synchrony of these events, have implications in synthesis of lactose has been reported by Bolander and Topper (1981). These investigators found that varying cortisol concentrations affected the alphalactalbumin content differentially from that of galactosyl transferase, activity of lactose synthetase, and secretion of lactose in explants from mature virgin mice cultured in medium containing insulin, prolactin and triiodothyronine

(T 3 ). The data indicated that galactosyltransferase activity better reflected lactose synthesis and secretion than did alphalactalbumin content. In a similar study, Ono et al.

(1981) demonstrated that hormonal regulation of casein

synthesis operates differently from that of alphalactalbumin. Mammary explants from midpregnant mice, cultured in the presence of insulin and prolactin displayed a cortisol dose-dependent inhibition of alphalactalbumin accumulation.

Conversely, casein synthesis increased with concentrations of cortisol. 26

Vonderhaar (1977), reported that alphalactalbumin activity in mouse mammary gland explants cultured in the presence of insulin, hydrocortisone and prolactin was increased 3 to

5 fold when (T 3 ) was further added to culture medium. This stimulatory effect of (T 3 ) was shown to be selective for alphalactalbumin synthesis by Terada and Oka (1982). These workers found that thyroid hormone synergizes with insulin, hydrocortisone and prolactin to elicit increased alphalactalbumin synthesis as well as higher levels of messenger RNA for alphalactalbumin, without appreciably affecting casein synthesis or casein mRNA accumulation in cultured mammary explants. The results of these studies have further demonstrated the complexity of the regulatory mechanisms governing synthesis of particular milk proteins. Bolander (1983) examined the requirements for insulin, cortisol and prolactin needed to induce casein synthesis and lactose synthetase activity in cultured mammary explants from virgin, pregnant, and nonlactating, nonpregnant, parous mice. Tissue from midpregnant mice was more responsive than that from virgins, requiring near-physiological hormone concentrations, in contrast with supraphysiological concentrations needed for vir- ginal tissue, to achieve induction of both casein synthesis and lactose synthetase activity. Tissue from parous mice responded to the lower dose of hormones for stimulation of 27

lactose synthetase activity, however induction of casein

synthesis required supraphysiological doses, similar to ex-

plants from virgins. These results illustrate that optimal hormone concentrations vary depending on the stage of gesta-

tion of the subject, as well as the biochemical endpoint se-

lected for measurement.

The above studies, in toto, demonstrate that hormonal re-

gulation of protein synthesis in the mammary epithelial cell

is indeed complex. That variation exists among hormonal ef-

fects on synthesis of various proteins, during various stag-

es of gestation, is clear. The secretory proteins alphalac-

talbumin and casein are apparently differentially regulated

by hormones in vitro. Thus serious implications are for-

warded on interpretation of degree of differentiation of

mammary tissue based upon specific endpoints or markers of

differentiation. It is important to realize that in a nor-

mal lactating gland protein synthesis occurs in synchrony,

but that individual protein levels may not be optimal by

standards of biochemical analysis. In spite of necessary caution, the accepted hormonal combination of insulin, cor-

ticoid and prolactin has a demonstrated capacity to stimu-

late lactogenic events to an extent comparable to parturient

lactogenesis. Measurement of secretory proteins in most

cases reliably reflects such lactogenic events, and as such 28

these measurements remain a logical and practical indicator of terminal differentiation of the mammary epithelial cell.

HORMONAL CONTROL OF LACTOGENESIS: RUMINANTS

By far the greatest proportion of in vitro experimenta- tion concerning hormonal regulation of lactogenesis has been conducted on mammary tissue from laboratory species. The results of these studies have been indicative of substantial variation between species in sensitivity to hormones. Thus the lactogenic complex, while being for the most part univ- ersal, also varies to a degree depending on the particular species in question. The existence of this variation made it clear that application of findings from small-animal studies to understanding of lactogenic events in ruminants was unacceptable without further research. To this end, much recent effort has been directed toward studying hormonal interactions involved in lactogenesis in ruminant mammary tissue, in vitro.

Jeulin-Bailly et al. (1973) cultured explants of midpregnant ovine mammary gland and used histological assessment to evaluate lactogenic response to added hormones. In an initial experiment, explants incubated for 10 days in medium supplemented with insulin alone, or combined with hydrocortisone, were well-maintained in terms of histological in- 29

tegrity. Presence of hydrocortisone apparently stimulated enlargement of alveolar lumina, but no secretion was visible. Addition of prolactin and growth hormone to the above combination did not result in morphological changes, however some stainable secretion was noted. A second phase of this study utilized a two day preculture with estrogen-progesterone plus either insulin and hydrocortisone, or insulin, hydrocortisone, prolactin and growth hormone, followed by 8 days of culture in the latter combination. Explants precul- tured in the presence of estrogen, progesterone, insulin and hydrocortisone displayed lobulo-alveolar development and evidence of secretory activity. Supplementation with the pituitary hormones during preculture elicited a further increase in secretory response after 10 days of culture.

Djiane et al. (1975) reported on the culture of mammary tissue from two- year old heifers. Tissue obtained was initially poorly developed, with only small ducts and end buds in evidence. Explants cultured in medium supplemented with insulin were well maintained, as were those exposed to insulin plus cortisol. Addition of prolactin to this combination caused explants to assume a secretory appearance, with evidence of active secretion. Further addition of ovine growth hormone did not augment secretory response. These researchers also discovered that a gas mixture of 57% 0 2 : 30

5% CO 2 : 38% N2 during incubation was superior to the 95% 0 2

: 5% CO 2 combination used almost exclusively in previous studies. These results suggest that in ruminants, prolactin supplies the requirement for a pituitary lactogen while growth hormone is without specific effect. Nickerson et al.

(1978) demonstrated that mammary explants from estrogen-progesterone primed heifers responded to stimulatory hormones in vitro. Tissue cultured in the presence of insulin and cortisol expressed histological evidence of lipid secretion and enlarged luminal area, although protein secretion was not observed. By contrast, explants exposed to insulin, cortisol and prolactin had more lipid, less stroma and fewer ducts, and displayed evidence of protein secretion. Addi- tion of estrogen and progesterone to this combination further stimulated secretory response. The authors proposed that ovarian steroids potentiated the effects of prolactin in stimulating differentiation of mammary epithelial cells.

Histology of insulin, cortisol, estrogen plus progesterone treated explants was well maintained, but did not assume a secretory appearance. These results, as well as those of

Jeulin-Bailly et al. (1973) illustrate the importance of estrogen and progesterone in lactogenesis. A second portion of this study (Nickerson et al., 1978) attempted to isolate highly lactogenic plasma samples obtained from cows during 31

various days of induced lactation. Plasma provided unknown stimulatory factors that enhanced differentiation of explants cultured in the presence of insulin, cortisol and prolactin. However explants responded similarly to all plasma samples, thus differential lactogenic merit could not be determined. The unexplained stimulatory effect of plasma is suggestive of the presence of as yet unknown factors or hormonal interactions which may be important components of the lactogenic complex. The above studies give histological evidence that insulin, corticoid and prolactin in combination provide the necessary stimuli to elicit lactogenic responses in mammary tissue from ruminants, in vitro.

Further study of hormonal regulation of lactogenesis in ruminants was aimed at describing control of biosynthesis of milk components. Collier et al. (1977) cultured mammary explants from pregnant cows and measured radioactive acetate incorporation into fatty acids in response to hormonal stimulation. The combination of insulin, cortisol and prolactin proved to be optimal in this respect. Explants from

60-day pregnant goats responded to incubation in the presence of insulin and cortisol with a four-fold increase in fatty acid synthesis and increased area occupied by epithelial cells (Forsyth and Turvey, 1984). Addition of ovine prolactin increased fatty acid synthesis another two-fold, 32

and secretory activity was elicited. Tissue from 120-day pregnant goats proved unresponsive to insulin plus cortisol, however supplemental prolactin maintained or slightly enhanced fatty acid synthesis and secretory activity, which was considerable at the start of culture. That hormones were able to stimulate fat synthesis supports in vitro study of lactogenesis as representative of in vivo events since increased milk-fat synthesis is normally associated with the onset of lactation. Such results must be tempered with the realization that fatty acid synthesis is not a specific indicator of terminal differentiation of mammary epithelial cells unless the carbon-chain length of the fatty acids is determined (Forsyth and Turvey, 1984).

The differential response of mammary tissue obtained during various stages of pregnancy reported by Forsyth and Tur- vey (1984) was noted earlier by Skarda et al. (1978). These investigators found that explants from glands of 2- or

24-day old goats were capable of secretory response during 6 days of culture in lactogenic hormones. However tissue from mature virgin and early pregnant animals remained unresponsive, even when cultured for 9-16 days. Ability to differentiate reappeared between 4 and 7 weeks of gestation and peaked at weeks 9-10. Surprisingly, explants from animals

12 or 18 weeks pregnant were again unresponsive. The au- 33

thors attributed lack of response by virgin and early pregnant tissues to a requirement for estrogen-progesterone sen- sitization of mammary tissue before prolactin could exert a stimulatory effect. Poor responses in explants from late pregnant goats was explained by low survival in vitro of highly differentiated mammary epithelial cells. Thus a tremendous differential in responsiveness to hormones exists between ruminant mammary tissues obtained at various stages of development. Similar results have been observed in mice

(Bolander, 1983).

Effects of hormones on protein synthesis by goat mammary tissue have been explored by Skarda et al. (1982a). These workers found that addition of prolactin to the combination of insulin and cortisol increased syntheses of casein and total protein by as much as 500 and 30%, respectively.

Further supplementation of the triple combination of hormones with (T 3 ) or progesterone had no apparent effect on prolactin-stimulated protein syntheses. The lack of synergism between (T 3 } and prolactin is in contrast with the findings of Vonderhaar (1977), in tissue from mice, and is indicative of species variation in response to T 3 • A subsequent study by this group examined the interrelationships involved in stimulation of protein, lipid and casein syntheses by insulin, prolactin and cortisol (Skarda et al., 34

1982b). Increased synthesis of each of the products was elicited during culture with insulin and prolactin, whether or not in the presence of cortisol. Results further indicated that prolactin and insulin acted independently in stimulating total protein synthesis, since their effects were addi- tive. However induction of casein synthesis was shown to be solely dependent on the presence of prolactin. These data led the authors to propose that while all of these hormones are involved in differentiation of mammary epithelia, prolactin is the primary stimulator of milk synthesis. Corti- sol apparently serves as an amplifier of prolactin-induced secretory function, however lack of response to added cortisol may reflect storage of this hormone at explantation, rather than lack of effect in vitro.

Goodman et al. (1983) conducted a study of the effects of various hormones on secretion of alphalactalbumin by mammary explants from 8 month pregnant cows. Cortisol was without effect when administered alone, but displayed the capacity to potentiate prolactin-stimulated alphalactalbumin secretion. By contrast, estradiol alone effectively enhanced secretion, and additionally synergized with prolactin to further increase secretion of alphalactalbumin. Progester- one alone increased secretion 2-fold over controls, however this hormone inhibited prolactin-associated stimulation, and 35

further interfered with synergism of estrogen with prolactin in promoting alphalactalbumin secretion. The conclusions of these researchers agreed with those arrived at by Skarda et al. (1982b) regarding prolactin as the essential promoter of mammary differentiation. However these investigators (Good- man et al., 1983) emphasized the importance of cortisol and estradiol interacting with prolactin, in the absence of progesterone inhibition, as the essential hormonal requirements involved in the onset of lactation.

Bovine growth hormone apparently has no direct effect on cultured mammary tissue from goats (Skarda et al., 1982c), or cows (Goodman et al., 1983). However both of these research groups reported that bovine growth hormone synergized with prolactin to increase protein synthetic responses above those obtained with prolactin alone. Thus growth hormone appears to function as a non-essential promoter of prolactin-induced differentiation in bovine mammary tissue, although the possibility that any stimulatory effect of growth hormone may be attributed to contamination of the hormone preparation with prolactin, cannot be dismissed.

Djiane et al. (1975) noted that explants of bovine mammary tissue were better maintained when incubated in an atmosphere of 57% 0 2 : 5% CO 2 : 38% N2 than those cultured under 95% 0 2 : 5% CO 2 . Forsyth and Turvey, (1984) confirmed 36

these results in tissue from goat mammary gland, in vitro.

These more recent results indicated that explants cultured in air were well-maintained, while 60% of those cultured in

95% 0 2 : 5% CO 2 showed evidence of degeneration, primarily in the form of sloughed epithelial cells. These explants also were poorly responsive to prolactin compared with those cultured in air. Such findings indicate that a 95% 0 2 : 5%

CO 2 atmosphere is suboptimal for in vitro studies of ruminant mammary tissue. Clearly it is important to define an optimal atmosphere for future studies of lactogenesis in vitro.

In light of the research cited above, it it apparent that insulin, corticoid and prolactin comprise the essential elements of the lactogenic complex in mammary tissue from ruminants, in vitro. Involvement of ovarian steroids, including a preparatory role of progesterone and a potentiating role of estrogens is also implicated. Findings have shown that lactogenic requirements among ruminants (including goats, sheep and cattle) are remarkably similar, indeed this similarity extends to monogastrics as well (Denamur, 1971; For- syth, 1971). Lactogenesis however, is a complex process and understanding of the involvement of hormones such as (T 3 ) and growth hormone, in addition to as yet unknown stimulatory factors in plasma, must be considered incomplete. Addi- 37

tionally, areas such as hormonal effec~s at the cellular and subcellular levels, as well as effects of gas phase on cultured tissue, are poorly understood. The progress of research on lactogenesis remains encouraging however, and yields promise that further research may answer such questions and contribute greatly to the understanding of lactation.

LACTATION IN MALE MAMMALS

It is common knowledge that male mammals, despite being endowed with mammary glands, do not lactate. Indeed, Daly

(1979) reviewed the physiological barriers to male lactation and concluded that changes in pre- and circumpubertal sexual differentiation, as well as an occurrence of lactogenic events analogous to those occurring in periparturient females, would be required for males to lactate. The unlike- lihood of such events, as well as a lack of evolutionary demand for lactation in males, would appear to ensure against any occurrence of male lactation.

In spite of these barriers, male lactation does occur, in fact it is not uncommon in some species. Nair et al. (1981) reported that spontaneous lactation is common in male goats.

These researchers studied two male goats that exhibited gynecomastia (mammary gland development in males) at 15 months 38

of age. Weekly milkings yielded up to 55 ml for one buck and 25 ml for the other. Both bucks were reproductively capable, having previously sired kids. Asdell et al. (1936) reviewed similar cases of lactation in male goats, citing in one instance yields of up to 750 ml of milk. Perhaps the earliest report of male lactation was recorded by Aristotle,

(1910 trans.) in which "a he-goat was milked by his dugs to such effect that cheese was made of the product." It is clear then that lactation does occur in male mammals, and that in the case of male goats, reproductive capability is not compromised.

Male lactation in species other than the goat has generally been associated with pathological states or hormone therapy. In human males, gynecomastia is often the result of an endocrine imbalance. Ingestion or overproduction of estrogens, as well as anti-androgenic medications may cause gynecomastia (Bercovici and Maudelonde, 1982). Turkington

(1972) reported that prolactin does not appear to be etiolo- gically related to development of gynecomastia, although pituitary disorders resulting in elevated prolactin concentrations in serum have been associated with development of this condition. In addition, Turkington (1972) proposed that a genetic predisposition toward gynecomastia, acting through increased sensitivity to hormonal stimuli, may exist. These 39

studies implicate estrogens and prolactin in abnormal male breast development, however such development cannot be

equated with lactation. Actual expression of milk from human males has been recorded by Huggins and Dao, (1954).

These researchers induced lactation in two men by treatment with luteotrophin (a prolactin-like hormone) following pro-

longed estrogen therapy for prostate cancer. Although these

findings are representative of abnormal hormonal status,

they clearly reflect the capacity for lactation of male mammary glands subjected to hormonal stimuli.

Terminal differentiation of mammary glands from males of

several species has been brought about by experimental hormonal manipulation. Turner and DeMoss (1934) obtained milk

from mammary glands of male cats following treatment with an

estrogenic compound (theelin) and corpus luteum extract fol-

lowed by injection of galactin (a crude pituitary prolactin

preparation). Male mice attained lobule-alveolar develop-

ment after treatment with theelin and corpus luteum extract

(Turner and Gomez, 1934a). Biochemical evidence of differ-

entiation of mammary glands from male mice treated with es-

trogen and progesterone in vivo was reported by Freeman and

Topper (1978a,b). These workers found alphalactalbumin ac-

tivity present in mammary tissue from treated mice, whereas

untreated tissue contained no detectable activity. Williams 40

and Turner (1961) induced lactation in a Holstein steer by injecting estradiol benzoate plus progesterone for 180 days.

Maximum secretion obtained was 15 ml per day. These investigators attributed poor mammary development in treated males to the relative lack of a mammary fat pad. In females, the fat pad occupies an area into which the ducts and lobule-alveolar system normally grow. Thus in the absence of a fat pad, the male gland is deprived of space required for extensive proliferation. Induction of lactation in male cattle has also been reported by Kiddy et al. (1965). Two castrated males received estrogen-progesterone treatment for a period sufficient to induce lacteal secretion. Secretion obtained varied from 1 to 3 ml per day, and milk composition in terms of total solids, fat and protein content was deemed essentially normal.

The above studies demonstrate that the physiological barriers to male lactation can be overcome through administration of appropriate hormonal stimuli. That secretion of milk has been obtained from males of different species, including ruminants and monogastrics, discourages the possibility that males of any particular species (for instance goats) are exceptionally sensitive to lactogenic hormones.

Study of lactation in males then, joins with research utilizing the techniques of induced lactation and organ cul- 41

ture of mammary tissue reviewed earlier, in providing essential tools for the study of lactogenesis. Availability of such methods encourages research concerned with the onset of lactation, and furnishes an optimistic outlook for resolving questions on various aspects of lactation as well as yielding promise of future increases in productivity of milk production animals. Chapter III

MATERIALS AND METHODS

ANIMALS

Twenty-four prepubertal animals, 6 to 8 months of age, were grouped by breed (Angus or Holstein) and sex with six

subjects in each breed by sex grouping. Subjects were fed hay ad libitum, supplemented with grain, and also received water ad libitum (see Angus males below).

Experiment I included the males of each breed. Holstein males were housed in box stalls and were placed on experiment between 9/6/83 and 10/31/83. Angus males were raised on fescue pasture with free access to their dams and were pretreated and slaughtered between 10/28/83 and 11/7/83.

Experiment II included females of both breeds. Angus females were housed in box stalls and received steroid injec-

tions between 9/12/83 and 11/18/83. Holstein females re-

ceived treatment between 1/27/84 and 2/6/84, and were housed

in metabolism crates in the Animal Sciences Building,

(VPI&SU) to avoid severely cold temperatures. Photoperiod

was maintained at 12L:12D to approximate the photoperiod to

which Angus females were exposed.

42 43

HORMONE PRETREATMENT OF SUBJECTS

All experimental subjects were pretreated with daily in-

jections of estradiol 17-~ plus progesterone (.1 and .25 mg/kg/day) for 7 days, followed by 7 days without injection.

Animals were slaughtered on day 15 after •initial injection.

Estradiol and progesterone (Sigma Chemical Co., St. Louis,

MO) were dissolved in benzylbenzoate (Sigma) over low heat

and mixed with corn oil to achieve a 20% benzylbenzoate so-

lution. The daily dose of steroids was administered in a

6-ml aliquot of this mixture via subcutaneous injection pos-

terior to the scapula (Smith and Schanbacher, 1973). Blood

samples were obtained by puncture of the coccygeal vessels

and were collected every 2 to 3 days throughout the treatment period.

ORGAN CULTURE OF MAMMARY EXPLANTS

Mammary glands were excised whole immediately after

slaughter, and kept warm in an insulated bucket while trans-

ported to the laboratory (approximately 5 to 15 min.). Mam-

mary parenchyrna was located in excised glands, removed and

cut into explant-sized pieces (3 to 4 mrn 3 ) under aseptic

conditions. Depending on the particular animal, relative

amount of parenchyrna vs stroma within explants varied. In

particular, explants from mammary glands of bulls of either 44

breed had greater proportions of stroma than did explants from heifers. Explants from each subject were rinsed twice in sterile medium 199 (Gibco, Grand Island, NY), and randomly allotted to culture dishes. Explant cultures were conducted in plastic culture dishes (#3037 Organ Culture Dish,

Falcon Plastics Co., Oxnard, CA) fitted with stainless steel grids (#3014 Organ Culture Grid, Falcon) which rested on the center well of the dish as described by Goodman et al.

(1983). One milliliter of appropriate medium was contained in the center well and 4 to 6 explants were placed on the grid within each culture dish. Explants were thus suspended at the medium-atmosphere interface inside the dish (Dils and

Forsyth, 1981). Dessication of medium was prevented by placing moistened ''O-ringstt around the center well of the dish. Dishes were placed in an incubator maintained at 37C and gassed continuously with 95% 0 2 : 5% CO 2 bubbled through a beaker of water inside the incubator. The 0 2 : CO 2 atmosphere was chosen over other possible gas mixtures, e.g., 0 2

: CO 2 : N2 or air, based on the success of Goodman et al.

(1983) in culturing bovine mammary explants under 95% 0 2 :

5% CO2. 45

INCUBATION MEDIUM

Medium utilized in these experiments was medium 199 containing Earle's salts, L-glutamine, sodium bicarbonate (26.2 mM), and 25 mM Hepes buffer, (Gibco). This medium was supplemented with sodium acetate (10 mM, Fischer Scientific

Co., Fair Lawn, NJ), and Antibiotic-Antimycotic solution

(Gibco), containing Penicillin G (100 Units/ml medium), So- dium Streptomycin Sulfate (100 ug/ml), and Fungizone (Ampho- tericin B, 2.5 ug/ml). In addition, bovine serum albumin

(BSA; fraction V, United States Biochemical Corp., Cleve- land, OH) was added at .2% w:v final concentration to pre- vent adsorption of proteins to plastic and glassware. Firtal culture media were prepared by further supplementing the above medium with hormones. Basal medium (B) included bovine insulin (Eli Lilly and Co., Indianapolis, IN; or Sig- ma), hydrocortisone (Sigma), and L-triiodothyronine (T 3 ;

Sigma) at final concentrations of 5 ug/ml, .5 ug/ml, and .65 ng/ml, respectively (Goodman et al., 1983). Stimulatory medium (P) was identical to B with the further addition of bovine prolactin (NIH-B6, National Institutes of Health,

Bethesda, MD) at 1 ug/ml final concentration. Medium 199 and all additives were combined, thoroughly mixed and the pH 46

adjusted to 7.3. Media were filter sterilized through Aero- disc filters (Gelman Sciences, Inc., Ann Arbor, MI), ~.45 micron pore-size, and stored refrigerated in sealed sterile bottles. Prior to dispensing into dishes media were warmed to 37C in the incubator. Media were prepared fresh as needed and each batch was used for no more than 10 days.

Media for tritiated ( 3 H-) protein labeling studies were prepared by aseptic addition of 3 H-amino acid mixture (#444K

Amersham, Arlington Heights, IL; 43Ci/mM) to sterile B or P medium to achieve a final concentration of 4 uCi/ml. Triti- ated medium was dispensed into wells of selected dishes in scheduled replacement of non-labeled medium (see incubation protocol below).

Determination of 3 H-thymidine incorporation into mammary cell deoxyribonucleic acid (DNA) employed medium 199 unsup- plemented except for the addition of 3 H-thymidine (#TRK-637

Amersham; 49Ci/mM) at a final concentration of 10 uCi/ml

(see protocol below).

INCUBATION PROTOCOL

Zero time was designated as the time at which explant cultures were placed into the incubator and was generally 30 to 45 min post-slaughter. Beginning at 24 h after zero time, media were aseptically harvested and replaced with 47

fresh media at 24-h intervals, thus media were collected at

24, 48, 72, and 96 h. Cultures ended at 96 h (after zero time). Harvested media were stored frozen (-20C) until as-

sayed for alpha-lactalbumin (Alac). The number of culture dishes incubated per animal was dependent upon the amount of mammary epithelium available to be explanted. Thus in the case of Holstein males, which exhibited the least glandular development, at least 12 cultures (six of each, Band P) were incubated. A total of 20 dishes (10 of each, Band P) were incubated in cases of plentiful epithelium as observed

in most females of both breeds.

Media supplemented with 3 H-amino acids were added in place of unlabeled media to two randomly selected dishes of each (Band P) treatment at the 24 to 48 and 72 to 96 h per-

iods. Cultures containing 3 H-amino acid-labeled media were

stopped after 24 h of incubation following introduction of

3 H-label. Both media and explants from these dishes were

collected and stored frozen (-20C), explants were blotted

dry on absorbent paper and weighed prior to freezing and la-

ter, homogenization.

Because some dishes (those receiving 3 H-label at 24 to 48 h) were stopped prior to 96 h of culture, dish numbers de-

clined over time. For example, 12 dishes per animal ini-

tially provided 12 media samples at 24 h; however since la- 48

beled cultures were a subset of the original 12, eight media samples plus four 3 H-media samples were collected at 48 h, eight media samples were harvested at 72 h, and four unlabeled plus four 3 H-labeled cultures were stopped and harvested at 96 h. Thus two data sets were compiled: 1) non- radiolabeled media, collected at 24, 48, 72, and 96 h, and

2) 3 H-labeled media (and explants), collected after 24 h of incubation in the presence of label, at 48 and 96 h. Non- labeled media were assayed for Alac, whereas media and homogenates of explants from cultures incubated in the presence of 3 H-amino acids were assayed for Alac, 3 H-labeled total protein (TP), and 3 H-labeled Alac, by radioimmunoassay

(RIA), trichloroacetic acid (TCA) precipitation and antibody

(Ab) precipitation of proteins, respectively (see methods below).

3 H-THYMIDINE INCORPORATION

Incorporation of 3 H-thyrnidine into mammary cell DNA was investigated by short-term (4 h) incubation of explants in medium 199 containing 3 H-thyrnidine at a final concentration of 10 uCi/ml. Mammary explants were blotted on absorbent paper and 200-300 mg were placed in 25 ml Erlenmeyer flasks containing 2.5 ml 3 H-thyrnidine labeled medium. Flasks were gassed with 95% 0 2 : 5% CO 2 , sealed and incubated at 37C in 49

a shaking water bath (GCA, Precision Scientific, Chicago,

IL). Following incubation, labeled medium was discarded and explants were rinsed 3 times in unlabeled medium 199, then stored frozen (-20C). Explants were homogenized using a Po- lytron tissue homogenizer (Brinkman Instruments, Westburg,

NY). Explants and 2 ml 1% BSA [diluted in phosphate buf- fered saline (PBS)] were combined in a plastic tube and homogenized with a 20 sec burst of the Polytron set at high speed. The Polytron tip was rinsed in a separate tube with

2 ml 1% BSA in a 10 sec burst. Rinse fluid was added to the homogenate tube and the rinse tube was flushed twice with 3 ml 10% TCA to remove adhering particles. Fluid from each rinse was added to the homogenate to yield 10 ml final vo-

lume. The resulting homogenate was centrifuged at 2000x g for 15 min and the supernatant was aspirated. The pellet was rinsed with 3 ml 10% TCA, recentrifuged and the supernatant discarded. The final pellet was dissolved using 2 ml

NCS tissue solubilizer (Amersham), with occasional vortex-

ing. Samples were assayed in duplicate at each of two volumes (50 and 100 ul) and added to 5 ml ACS II scintillation cocktail (Amersham) in glass minivials. Radioactivity was determined by liquid scintillation spectrophotometry using a

Beckman spectrophotometer (Beckman Instruments, Fullerton,

CA). Incorporation of 3 H-thymidine could not be determined

for several subjects due to lack of mammary parenchyma. 50

3 H-PROTEIN QUANTIFICATION

Incorporation of 3 H-amino acids into TP and Alac fractions was determined in media and homogenates of explants

recovered from cultures incubated in the presence of

3 H-label. Homogenization of 3 H-labeled explants was accom- plished by combining explants and 400 ul .05% sodium dodecyl

sulfate (SOS) : PBS in a Duall glass to glass tissue grinder

(Kontes, Vineland, NJ) and homogenizing with 30 strokes.

The homogenate was decanted into a collection vial, the tis-

sue grinder rinsed twice with 300 ul PBS and the rinse added to the sample to yield 1 ml of homogenate. The homogenate was centrifuged at 2000x g for 30 min and the resulting supernatant was aspirated using a pasteur pipette and stored frozen (-20C) until assayed. Tritium-labeled TP was determined in media and homogenate samples by TCA precipitation of proteins. Each sample was assayed in triplicate. Sam- ples were prepared in 50 ul aliquots added to 200 ul 1% BSA

in a plastic minivial. Three ml ice cold TCA (10%) were added and minivials were placed in an ice bath for 10 min to

allow proteins to precipitate. Samples were centrifuged at

4000x g for 30 min and the supernatant aspirated and discarded. Pellets were solubilized by adding 500 ul NCS (Am- ersham) and allowing the pellet to dissolve overnight at

room temperature. Five milliliters of ACS II scintillation 51

cocktail (Amersham) were added to each sample vial and radioactivity determined by liquid scintillation spectrophotometry.

Determination of 3 H-Alac in media and homogenates was by double-antibody precipitation of Alac. Sample preparation was identical to that for 3 H-TP determination (above). Al- phalactalbumin in samples was complexed by incubation of samples with 100 ul rabbit anti-bovine Alac antibody (rabbit serum; 1:100 dilution) for approximately 24 hat room temperature. The antibody-Alac complexes were precipitated by addition of 100 ul ovine anti-rabbit gamma globulin serum

(1:19 dilution) followed by incubation for 24 hat 4C. Pel- lets were formed by addition of 3 ml cold PBS and centrifu- gation at 3500 x g for 30 min. The resulting supernatant was aspirated from the minivial and discarded. Solubiliza- tion of the pellet and determinatio,n of radioactivity was as described for quantification of 3 H-TP (above). Recoveries of 3 H-TP and 3 H-Alac precipitations were 85 to 94% and 68 to

74% efficient, respectively, based on characterization of assays employed (see Appendix a). Briefly, varying volumes of 125 !-Alac (representing various concentrations of protein) were combined with 1% BSA-PBS to yield 250 ul final volume, and total radioactivity was determined in each sample using a Gamma 4000 spectrophotometer (Beckman). These 52

samples were precipitated according to the above protocols, recovered radioactivity determined and recovery efficiencies

(cpm precipitated/cpm originally in sample) were calculated for each sample.

Measurement of 3 H-proteins (TP and Alac) in media and homogenates was intended to provide an indication of secretion versus synthesis of proteins. Unlabeled proteins in media or homogenates may have been synthesized and/or secreted at any point prior to, or during culture. However labeled proteins recovered from homogenates must have been synthesized within a given 24 h period and 3 H-proteins recovered from media must have been synthesized and secreted within the same period. Thus determination of timing and coordination of synthesis and secretion was possible.

PROTEIN RADIOIMMUNOASSAYS

Quantification of non-radiolabeled Alac in media, explant homogenates, and serum samples was by double-antibody radioimmunoassay (RIA) as described by Akers, et al. (1984).

Concentrations of prolactin in serum were determined by double-antibody RIA as described by Koprowski and Tucker,

(1971). 53

STATISTICAL METHODS

Data from all experiments were analyzed by analysis of variance. Because the major interest of the study was in breed differences, least squares means for breeds at each treatment x period level were compared using orthogonal contrasts (McGilliard, 1984). Analysis of concentrations of

Alac in medium at 24, 48, 72, and 96 h were performed on means of all dishes from each breed· x treatment x period group. All other analyses were based on individual dish means. Quantities of non-labeled Alac, 3 H-TP and 3 H-Alac in media and homogenates were analyzed using the following model: Breed, Animal(Breed), Treatment, Breed x Treatment, An- imal(Breed) x Treatment, Period, Breed x Period, Ani- mal(Breed) x Period, Treatment x Period, Breed x Treatment x

Period, Residual. Mean squares associated with Breed were tested by those from Animal(Breed); Treatment and Breed x

Treatment were tested by Animal(Breed) x Treatment; Period and Breed x Period were tested by Animal(Breed) x Period.

The remaining effects and interactions were tested by the residual mean squares. Representative ANOVA tables are seen in Appendix b. Chapter IV

RESULTS

EXPERIMENT I. MALES

Media Alpha-lactalbumin

Least squares mean concentrations of Alac secreted into medium by mammary explants from beef and dairy bulls are depicted in figure 1. Explants from prepubertal bulls of both breeds clearly demonstrated the capacity to secrete

Alac in response to the culture regimen imposed. However,

secretory patterns differed between the breeds. At 24 h, explants from Holstein bulls cultured in the presence of B or P secreted greater concentrations of Alac into medium than did corresponding cultures of Angus mammary tissue.

This relationship was reversed at the 48 hand ensuing periods, during which times explant cultures from Angus bulls proved more productive than those from Holsteins. Although these differences did not result in a significant overall effect of breed (p>.10), comparison of breed means within each treatment x period combination revealed that explants

from Angus bulls cultured in P secreted significantly great-

er concentrations of Alac at 48 h (382.6 vs 140.2) and 72 h

(500.3 vs 221.9 pg/mg/ml/24 h) than similarly-treated explants from Holstein bulls (p<.01).

54 55

TAT LEGEN~ TRT -AB -AP c::::J HB c::::J HP

Figure 1: Secretion of Alpha-lactalbumin into medium by mammary explants from Angus and Holstein bulls.

Units are in pg/mg tissue/ml medium/24 h. Standard error of means (SEM) = 60.6. *Greater (p<.01) than corresponding mean for Holstein. aTime of incubation. bin this and following figures: AB= Angus, basal medium. AP= Angus, stimulatory medium. HB = Holstein, basal. HP= Holstein, stimulatory. 56

The stimulatory effect of prolactin on secretion of Alac from mammary explants of young bulls is also illustrated in figure 1. Mean Alac secretion from cultures containing P was greater in both breeds at all time periods than secretion from B treated cultures. Thus presence of prolactin in medium resulted in increased secretion of Alac (p<.005).

3 H-Total Protein and Alpha-lactalbumin in Media

Incorporation of 3 H-amino acids into total protein (TP) and 3 H-Alac fractions by cultured mammary explants from beef and dairy bulls during the 24-48 and 72-96 h incubation periods is summarized in figures 2 and 3. Because these measurements were made in medium, newly formed proteins must have been synthesized and secreted during the respective time periods. Least squares mean counts per minute detected in TP were greater overall in cultures of Angus tissue than in those of Holstein tissue (pS.01). In addition, mean cpm recovered in TP from cultures of Angus explants were greater than those from Holsteins within each of the treatment x period groups (p<.005).

Determinations of 3 H-TP and 3 H-Alac in media were from aliquots of identical media samples, thus mean cpm detected in Alac fractions represented a proportion of the mean cpm recovered in TP fractions. Mean cpm detected in Alac recov- 57

TRT LEGEND: TRT -RB -AP C=:JHB C=:J HP

3 Figure 2: Secretion of H-Total Protein into media by Angus and Holstein bull explants.

Units are in cpm/mg/ml/24 h. SEM = 41.9 **Greater (p=.0001) than corresponding Holstein mean. Greater (p<.005) than corresponding Holstein mean. 58

TAT LEGEND: TRT -RB -RP c:::::J HB c:::::J HP

Figure 3: Secretion of 3 H-Alpha-lactalbumin into media by Angus and Holstein bull explants.

Units in cpm/mg/ml/24 h. SEM=l7.4 *Greater (p<.05) than corresponding Holstein mean. ** Greater (p<.10) than corresponding Holstein mean. 59

ered from media followed the same general pattern as 3 H-TP.

Cultures of male Angus tissue accumulated greater amounts of

3 H-Alac in medium overall than did Holstein bull cultures

(p<.05), as well as in both of the treatments at each period

(p=.07-.0001). Relative proportions of 3 H-TP consisting of

3 H-Alac ranged from 31.5-41.0% in each of the treatment x period combinations for Angus males, and averaged 37.1% overall, compared with 47.3-58.9% and 50.8% overall for Hol- stein males. Thus Alac apparently made up a greater proportion of newly synthesized total secretory protein in

Holstein cultures as compared to Angus cultures, although this relationship was not tested statistically. Total quantities of radiolabeled Alac in media were greater at 96 h compared with quantities detected at 48 h (pS.05).

3 H-Total Protein and Alpha-lactalbumin in Homogenates

Quantities of 3 H-TP and 3 H-Alac detected in homogenates of explants following incubation from 24-48 and 72-96 h in the presence of 3 H-amino acids are illustrated in figures 4 and 5. Homogenate values represent the portion of TP and

Alac synthesized but not secreted during each 24 h incubation in the presence of 3 H label. As seen in figure 4, mean accumulation of TP in explants from Holstein bulls at 48 h was greater, averaging 63.1 and 77.2 cprn/rng/ml/24 h for B 60

92. 4.*

77. 2*

TRT LEGEND: TAT -RB -RP c::::J HB c::::J HP

Figure 4: Concentrations of 3 H-Total Protein in homogenates of Angus and Holstein bull explants

Units in cpm/mg/ml/24 h. SEM = 6. 7 *Greater (p<.005) than corresponding Angus mean. 61

HOURS

9. l!*

TRT LEGEND: TAT -AB -AP [=:lHB c::::J HP

Figure 5: Concentrations of 3 H-Alpha-lactalbumin in homogenates of Angus and Holstein bull explants.

Units in cpm/mg/ml./24 h. SEM = .6 *Greater (p=.0001) than corresponding Angus mean. ** Greater (p•.005) than corresponding Angus mean. 62

and P treated cultures compared with 29.3 and 38.8 cpm/mg/ ml/24 h in explants from Angus bulls at comparable time and treatment (p<.001). Breed means did not differ significantly at 96 h among cultures receiving B, however in the presence of P, Holstein bull explants accumulated more (92.4 vs

62.1 cpm/mg/ml/24 h) labeled TP than those from Angus males

(p<.005). Presence of prolactin had an overall stimulatory effect on synthesis of TP (p<.05). Total mean 3 H-TP (combined over breed and period) recovered from P treated cultures was 270.5 cpm/mg/ml/24 h compared with 188.0 cpm/mg/ ml/24 h from B treated cultures. Mean accumulation of 3 H-TP was also greater in P treated explants of both breeds at each of the periods, than in cultures exposed to B (p<.05).

Least squares mean concentrations of 3 H-Alac in explant homogenates from Holstein bull cultures compared with those from Angus bulls were 8.3 vs 3.5, and 9.4 vs 4.2 for Band P at 48 h, and 6.0 vs 3.5, and 9.0 vs 4.0 cpm/mg/ml/24 h for B and P medium at 96 h, respectively. In each case, Holstein bull explants accumulated more 3 H-Alac than did corresponding Angus bull explants (pS.005). Relative percentage of

3 H-TP comprised of 3 H-Alac in explant homogenates from Hol- stein bulls ranged from 9.7 to 13.2% during the various time x treatment groups and averaged 12.0% over all times and treatments. Tritiated Alac as a percentage of 3 H-TP in horn- 63

ogenates ranged from 6.4 to 11.9% and averaged 9.0% overall

in explants of Angus bull mammary tissue. Although not tested statistically, 3 H-Alac made up a greater percentage of newly synthesized secretory protein during each time and treatment in explant homogenates from Holstein bulls compared to those from Angus bulls. This relationship was similar to that observed in media samples from these animals

(see figures ·2 and 3). Explants cultured in the presence of

P accumulated more 3 H-Alac at each of the breed x period

combinations than explants exposed to B. Thus over both breed and period, presence of prolactin tended to enhance

3 H-Alac accumulation (p=.07), similar to the stimulatory ef-

fect of prolactin on 3 H-Alac accumulation in media (figures

2 and 3).

Non-radiolabeled Alpha-lactalbumin Concentrations in Media and Explant Homogenates

Least squares mean concentrations of Alac secreted into

medium or retained within explants of mammary tissue from

beef and dairy bulls incubated in the presence of B or Pat

24-48 and 72-96 hare portrayed in figures 6 and 7. Mea-

surements of Alac for these figures were obtained using ali-

quots of media and homogenates from samples used to quantify

3 H-TP and 3 H-Alac in media and homogenates as depicted in

figures 2-5. Because Alac concentrations in figures 6 and 7 64

TRT LEGEND: TRT · -RB -RP c::::J HB c::::J HP

Figure 6: Secretion of non-radiolabeled Alpha-lactalbumin into media by bull explants exposed to 3 H-amino acids during culture.

Units in pg/mg/ml/24 h. SEM • 37 .3 *Greater (p<.005) than corresponding Holstein mean. 65

TRT LEGEND: TRT -AB -AP c=:J HB c=:J HP

Figure 7: Concentrations of non-radiolabeled Alac in homogenates of bull explants exposed to 3 H-amino acids during culture.

Units in pg/mg/ml/24 h. SEM"" 16.6 *Greater (p<.0005) than corresponding Holstein mean. ** Greater (p=.05) than corresponding Holstein mean. 66

were quantified by RIA rather than 3 H-amino acid incorporation, values may represent protein synthesized at any time prior to the sampling period. Thus carryover effects of protein synthesis occurring at earlier time periods or even in vivo (prior to explantation) may be reflected in these data.

As shown in figure 6, Angus bull explants tended to secrete greater quantities of Alac into medium overall, than did explants from Holstein bulls (p=.10). Comparison of breed means within each period x treatment group indicated greater secretion of Alac by Angus explants cultured in Pat

96 h, compared with mean secretion from comparable Holstein explants (p<.dOS). Overall analysis of treatment effect suggested a stimulatory effect of Pon secretion {pS.10).

Secretion of Alac into medium was increased at 96 vs 48 h in each of the breed x treatment combinations although these means were not compared statistically. However, total mean secretion (over all breed and treatments combined) proved greater at 96 than at 48 h (176.1 vs 99.2 pg/mg/ml/24 h; pS.01).

Concentrations of Alac present in explant homogenates from bulls of both breeds at the indicated time and treatment levels are depicted in figure 7. Alpha-lactalbumin concentrations in Angus explants averaged 173.0 and 198.9 67

pg/mg/ml/24 h for Band P treated cultures at 48 h compared to 84.5 and 81.9 pg/mg/ml/24 h in comparable cultures of

Holstein explants. In each of these cases explants from An- gus bulls contained greater concentrations of Alac than those from Holstein bulls (p<.0005). Mean concentrations of

Alac were also elevated (pS.05) in Angus explants exposed to

Bat 96 h compared with similarly treated explants from male

Holsteins (117.9 vs 71.1 pg/mg/ml/24 h). In addition, an overall tendency for increased concentrations of Alac in An- gus explants was observed (pS.10). Explants cultured in P accumulated an average of 149.4 pg/mg/ml/24 hover both breeds and periods, whereas the overall mean of B-treated explants was 111.6 pg/mg/ml/24 h. Thus a significant overall effect of treatment existed (p<.05). Means also tended to differ between breed x treatment x period groupings

(p<.10).

Synthesis versus Secretion of Proteins~ Cultured Mammary Explants from Angus and Holstein Bulls

Least squares means for overall synthesis and percent

secretion of TP and Alac in cultured mammary explants from dairy and beef bulls are presented in table 1. Values reported for mean total synthesis represent the mean quantities of TP or Alac detected in medium and homogenate combined to yield a total synthesis measurement for each breed 68

TABLE 1 Synthesis and secretion of milk proteins by cultured mammary explants from Angus and Holstein bulls.

I 11 Time .Q.f culture (h) __ I 11 11 2-2 I I BxT:l I I Total I Percent I I Total I Percent IParameterlGroupl I Synthesis I Secreted! I Synthesis I Secreted I I I 11 . I I AB I 526. 8,1<. I 93_3* I I 446. 9** I 87.6* I HB I 203.8 I 68.2 11 262.7 I 75.6 3 H-TP~ I I 11 I I AP 421.5* I as.a* I 587. s·"~ I 88.7~ I HP 174.9 I 63.5 I 431.1 I 74.7 I I I I AB 196. 9* I 97.8* I 151. dH~1 97.1* HB 108.6 b I 91.2 I 85.3 I I 86.6 3 H-Alac I I I ~I I AP 125.3* I 9s.o* I 219. 4"'~ · 1 98.1.\(. I HP 57.5 I 82.9 I 171. 3 I 87.9 I I I I I AB 270. cf.,:1 38.1 I 249.7 I 55.5 HB 137.8 39.l 204.2 46.2 C. I I I I Alac I I I "--t. I I AP 360. 1**1 41.0 I 452 .1· I 65. 7~ I HP 167.3 I 47.8 I 304.2 I 38.8

~Greater (p<.0005} than corresponding mean of opposite breed. **Greater (p<.05} than corresponding mean of opposite breed. ~**Greater (p~.10) than corresponding mean of opposite breed. aCpm/mg/ml; SEM for total synthesis= 43.0, for percent secreted= 1.9. bCpm/mg/ml; SEM for total synthesis= 18.7, for percent secreted= .9. cPg/mg/ml; SEM for total synthesis= 45.2, for percent <1secreted = 4.7. BxT = Breed by Treatment. 69

x treatment x period group (ie: synthesis= media+ homogenate). Mean secretory percent was determined by calculating the proportion of total synthesis contributed by proteins recovered from media (ie: secretory percent= media/ total

synthesis).

Total synthesis of 3 H-TP in Angus bull explants averaged

526.8, 421.5, for Band Pat 48 h; and 446.9, 587.8 cpm/mg/ ml/24 h for Band Pat 96 h compared with 203.8, 174.9, for

Band Pat 48 h; and 262.7, 431.1 cpm/mg/ml/24 h for Band P

at 96 h, respectively, in Holstein explants. In each case

Angus bull explants synthesized greater quantities of TP than did their Holstein counterparts (p<.01). Breeds also differed in total 3 H-TP synthesis combined over both times

and treatments (p<.05). In addition, explants of male Angus mammary tissue secreted a greater percentage of newly synthesized TP than did Holstein explants. Mean secretory percent for cultures of Angus explants ranged from 85.8 to

93.3% across the four treatment x period groups compared with 63.5 to 75.6% secretion by Holstein cultures. Each of these treatment x period means were greater in Angus than in

Holstein cultures (p<.0005). The effect of Pon mean total

synthesis and secretory percent in both breeds differed between 48 and 96 h, yielding a significant treatment x period

term in the analysis (p<.05). 70

Cumulative synthesis of 3 H-Alac was increased in Angus explants at 48 h, averaging 196.8 and 125.3 cpm/mg/ml/24 h

for Band P treated cultures, compared with 91.2 and 57.5 cpm/mg/ml/24 h, respectively, in Holstein explants (pS.01).

Relative differences between breeds within each treatment were less pronounced at 96 h, however the tendency for greater synthesis by Angus explants remained (p<.10). Ove-

rall effects of breed (pS.05) and period (p<.10) were indi-

cated by statistical analyses, as was a difference in treatment effect over time (pS.0001). Mean percent of 3 H-Alac

secreted was greater in Angus explants but remarkably simi-

lar within explants of each breed, ranging from 95.0 to

98.1% in Angus explants and 82.9-87.9% in Holstein explants

over the four treatment x period groups (pS.0001). Angus bull cultures also tended to synthesize and secrete more

3 H-Alac over all time and treatments combined than did cul-

tures of Holstein bull tissue (pS.10). Like total synthesis

of 3 H-Alac, effect of treatment on mean secretory percent was dependent on incubation period (p<.005), with prolactin exerting a stimulatory effect only at 96 h. Thus Angus bull

explants synthesized greater amounts and secreted higher

percentages of both 3 H-TP and 3 H-Alac within each treatment

x period. 71

Cumulative synthesis and total percent secreted of non radiolabeled Alac cannot be directly compared to measure 3 ments of H protein fractions, although all parameters were measured in aliquots of identical samples. Because RIA es timates of Alac concentrations did not differentiate time of synthesis, Alac accumulated may represent protein synthes ized during an earlier incubation period. In spite of these differences, cumulative Alac synthesis 3 followed a pattern similar to that of H-Alac synthesis. Angus explants accumulated greater quantities of Alac in medium and homogenate combined, in cultures exposed to B or Pat 48 h and in cultures exposed to Pat 96 h, averaging 270.0, 360.1, and 452.1 pg/mg/ml/24 h compared with 137.8, 167.3, and 304.2 pg/mg/ml/24 h respectively in Holstein cul tures comparably treated (p<.05). Accumulation of Alac to talled over both treatments and periods was increased in An gus explants as well (p<.01). Presence of prolactin in medium had a stimulatory effect on overall accumulation of Alac across both breeds and per iods (p<.05). Percent of Alac secreted into medium was greater in P-treated cultures of Angus explants at 96 h, av eraging 65.7% compared with 38.8% from the corresponding Holstein cultures (p<.0005). However breed differences were not apparent in the remaining time x treatment groups (p>.10). 72

Secretory percent also tended to increase from 48 to 96 h

(p<.10), although this relationship was affected by breed

(breed x period,p~.05), and treatment (breed x treatment x period, p<.10) influences.

Correlations

Analyses of the relationships between various parameters revealed large correlations between quantities of 3 H-TP and

3 H-Alac recovered from media and homogenates. Among Angus bull explant cultures, media concentrations of 3 H-TP were correlated with _3 H-Alac precipitated from media (r=.91), as were quantities detected in homogenates (r=.50). Holstein bull cultures exhibited similar characteristics, with strong correlations evident between 3 H-TP and 3 H-Alac concentrations in media (r=.86), and in homogenates (r=.95), respectively. Correlation coefficients for other pairs of parameters are presented in Appendix c.

3 H-Thymidine Incorporation into Mammary Explant DNA

Incorporation of 3 H-thymidine into DNA at zero time in mammary tissue explants from Angus and Holstein bulls is illustrated in table 2. Mammary explants from Holstein bulls incorporated an average of 139.2 cpm/mg/h compared to 107.9 cpm/mg/h in explants from Angus bulls. Rates of incorpora- 73

TABLE 2

Incorporation of 3 H-thymidine into DNA of mammary explants from Angus and Holstein bulls.

a I Breed I N !Mean CPM S.E.M. I I I I 1-1 I Angus (A) I Sb I 107.9 34.1 I I I !Holstein (H) I 6 I 139.2 31.1 I I I

aLeast squares mean (cpm/mg/h) 3 H-thymidine incorporation, ± standard error of mean. bone Angus bull lacked sufficient mammary tissue for this analysis after assignment of explants to culture. 74

tion were not statistically different between breeds

(p>.10).

Serum Prolactin and Alpha-lactalbumin

Concentrations of prolactin in serum samples from Angus and Holstein bulls during steroid pretreatment therapy are summarized in table 3. Mean prolactin concentrations in serum from Holstein bulls were increased by a factor of two over those detected in Angus serum during the 15 day pretreatment period (p<.05). No difference in prolactin concentrations was observed between the injection (days 1-7) and post-injection (days 8-15) periods in bulls of either breed (p>.10).

In addition to prolactin concentrations, serum samples were also assayed for Alac content. Efforts to detect Alac in serum were largely unsuccessful. Concentrations of Alac in serum from bulls of both breeds were generally undetectable.

EXPERIMENT II. FEMALES

Media Alpha-lactalbumin

Concentrations of Alac detected in culture media follow-

ing incubation of mammary tissue from Angus and Holstein heifers are shown in figure 8. during the initial 24 h of 75

TABLE 3 Concentrations of prolactin in serum of Angus and Holstein bulls during steroid pretreatment.

I I Mean I Breed I Daysa I Nb I Prolactirfl S.E.M. I I I I I 1-1 I Angus (A) I 1-7 (I) I 34 I 5.6 I .9 I 8-15 (PI) I 48 I 5.1 I 1.1 I I I I Holstein (H) I 1-7 (I) I 31 I 10.1* I 1. 7 I 8-15 (PI)I 37 I 13. 6-1( I 1.5 I I I I

aSamples collected during-, (I), or post-injection (PI) of estradiol and progesterone. bsarnples were collected randomly throughout the pretreatment period. Not every subject was sampled on a given day. cMean concentrations of serum prolactin (ng/ml), ± standard error of mean. *Greater (p<.05) than corresponding mean for Angus. 76

HOURS 96 B 119 152

32.5

TIIT LEGE NO: TR.T -RB -AP c::J HB c:::J HP

Figure 8: Secretion of Alpha-lactalbumin into medium by mammary explants from Angus and Holstein heifers.

Units in pg/mg/ml/24 h. SEM = 103.9 *Greater (p<.0005) than corresponding Angus mean. ** Greater (p•.10) than corresponding Angus mean. Note: mean bars for RB and HP at 24 hare attenuated, numeric means are correct. 77

incubation, Holstein explants cultured in B or P secreted 30 to 40-fold greater concentrations of Alac into media than similarly treated Angus explants (pS.0001). Secretion of

Alac from Holstein explants decreased between 24 and 48 h, however the difference between breeds remained apparent among those cultures exposed to prolactin, as P-treated Hol- stein explants continued to secrete 30 times more Alac than corresponding explants from Angus heifers (p<.0005). At 72 h, P-treated Holstein explants still tended to secrete greater quantities of Alac than comparable Angus explants

(pS.10), however, the relative difference between breed means was decreased to approximately four-fold. Rates of Alac secretion did not differ between breeds among cultures exposed to Bat 48 through 96 h of incubation, nor within P- treated cultures at 96 h (p>.10). Overall mean secretion of

Alac declined during the 96 h of incubation (p<.05), although Angus cultures maintained their initial secretory rates to a greater extent than did cultures of Holstein explants (p<.05). Presence of prolactin in medium apparently magnified the difference in Alac secretion between breeds, however, no overall effect of treatment was observed

(p>.10). 78

TRT LEGEND: TRT -AB -AP c:=)HB c=:JHP

Figure 9: Secretion of 3 H-Total Protein into media by Angus and Holstein heifer explants.

Units in cpm/mg/ml./24 h. SEM = 17.4 *Greater (p<-.005) than corresponding Angus mean. ** Greater (p•.07) than corresponding Angus mean. 79

TRT LEGEND: TRT -AB -AP c::J HB c::J HP

Figure 10: Secretion of 3 H-Alpha-lactalbumin into media by Angus and Holstein heifer explants.

Units in cpm/mg/ml/24 h. SEM = 10.9 *Greater {p~.01)- than corresponding Angus mean. ** Greater (p=.06) than corresponding Angus mean. 80

3 H-Total Protein and Alpha-lactalbumin in Media

Incorporation of 3 H-amino acids into 3 H-TP and 3 H-Alac fractions recovered from media following culture of mammary explants from Holstein and Angus heifers is illustrated in figures 9 and 10. As seen in figure 9, Holstein explants synthesized and secreted greater quantities of TP at 48 h than did Angus explants, averaging 143.5 and 143.1 cpm/mg/ ml/24 h from Band P treated cultures, compared with 60.6 and 68.5 cpm/mg/ml/24 h from comparable Angus cultures (p<.005). Holstein explants cultured in Palso tended to produce more 3 H-TP at 96 h than similarly-treated Angus explants {p<.10), although B-treated explants did not differ by breed at this time (p>.10). Overall concentrations of

3 H-TP in media (all treatments and periods combined) tended to be increased in Holstein cultures as well (p<.10). Pro- lactin apparently did not stimulate synthesis and secretion of 3 H-TP in these cultures, (p>.10).

Synthesis and secretion of 3 H-Alac tended to be increased (p<.10) in explants from Holstein heifers above those from Angus, averaging 63.1 vs 2~.7 cpm/mg/ml/24 hover all treatments and periods combined {figure 10). Holstein explants were more productive at 48 h than Angus explants, averaging

three-fold greater concentrations of 3 H-Alac in both B- and 81

P-treated cultures (p<.01). Secretion of 3 H-Alac was approximately two-fold greater in Holstein cultures exposed to

Band Pat 96 h compared with that from corresponding Angus cultures (pS.06). Mean concentrations of 3 H-Alac in media for each breed were similar across both treatments and periods (p>.10), revealing a lack of prolactin stimulation and no detrimental effects of time in culture on activity of explants.

Concentrations of 3 H-Alac in media represent a proportion of total 3 H-protein ( 3 H-TP) recovered from media. Because they were measured in aliquots of identical media samples,

3 H-Alac as a percentage of 3 H-TP could be determined.

Counts per minute detected in 3 H-Alac made up 44.1-56.4% of total counts recovered in precipitated protein from Holstein cultures and averaged 48.7 ± 5.7% (mean± sd.) overall, compared with 30.0-36.2% (mean= 33.1 ± 3.3%) from Angus cultures exposed to the various treatment and period levels.

Thus Alac appeared to contribute a higher percentage of secretory protein in explant cultures of Holstein tissue than in those of Angus explants, although this difference was not tested statistically. 82

HOURS

153.s* 133.

48 * 114. 9

TRT LEGEND: TAT -RB -RP c:=iHB c:=i HP

Figure 11: Concentrations of 3 H-Total Protein in homogenates of Angus and Holstein heifer explants.

Units in cpm/mg/ml/24 h. SEM = 6.1 *Greater (p=.0001) than corresponding. Angus mean. 83

3 H-Total Protein and Alpha-lactalbumin in Explant Homogenates

Concentrations of 3 H-TP and 3 H-Alac precipitated from homogenates of Angus and Holstein explants following incubation for the indicated time and treatment are presented in figures 11 and 12. As seen in figure 11, synthesis of 3 H-TP was increased in cultured Holstein explants over that in An- gus explants for each of the treatment by period groups

(pS.0001). Synthesis of TP combined over treatments and periods averaged 131.0 and 47.4 cpm/mg/ml/24 h for Holstein and Angus explants, respectively (p<.01). Accumulation of

3 H-TP in Holstein explants increased from 48 to 96 h in contrast to the decrease in TP observed over time in Angus explants (p<.005). The effect of presence of prolactin in me-

dia also differed between these breeds (p<.01). Angus

explants exposed to P synthesized more TP at each period

than those exposed to B, whereas Holstein explants were ap-

parently not stimulated by P.

That synthesis of 3 H-Alac by explants reflected the pat-

tern observed for production of TP is demonstrated in figure

12. Holstein explants accumulated more 3 H-Alac in each of

the treatment x period groups than their Angus counterparts

(pS.0001). In addition, overall mean synthesis of 3 H-Alac

was increased in Holstein cultures compared to that occur- 84

HOURS

12.2 *

TRT LEGEND: TAT -AB -AP c:J HB c:J HP

Figure 12: Concentrations of 3 H-Alpha-lactalburnin in homogenates of Angus and Holstein heifer explants.

Units in cpm/mg/ml/24 h. SEM ... 7 *Greater (p•.0001) than corresponding Angus mean. 85

ring in Angus cultures (pS.005). Similar to 3 H-TP, Alac synthesis increased over time in Holstein explants, while

Angus explants became less productive as time progressed

(p<.005). Response of explants to treatment imposed was again different between breeds, with Angus but not Holstein cultures producing more Alac in the presence of prolactin

(p<.001).

Tritiated Alac as a percent of 3 H-TP present in homogenates was remarkably similar across breeds. Within Angus explants, 11.0-12.2%, (11.7 ± .7% mean± sd.) of the cpm detected in protein was contributed by Alac. Homogenized

Holstein explants contained 10.6-11.7% Alac, which averaged to 11.2±.5%. Thus 3 H-Alac made up a nearly constant proportion of 3 H-TP in homogenates of Angus and Holstein explants, in contrast to the increased percentage of 3 H-Alac in media from Holstein cultures (see figures 9 and 10).

Non-radiolabeled Alpha-lactalbumin in Media and Homogenates

Mammary explants from Holstein heifers secreted greater

(p<.05) concentrations of non-radiolabeled Alac into culture media than explants from Angus heifers (figure 13). Indivi- dual breed means for each treatment and period level were greater in Holsteins (pS.0001), with the exception of P- treated explants at 48 h, which did not differ by breed 86

(p>.10). Secretion of Alac tended to increase between 48 and 96 h (pS.10) in both breeds. B~eed, treatment and period effects interacted significantly (p<.10) as reflected by the relatively larger increase in Alac secretion by Holstein explants exposed to Pat 96 h.

Breed differences were also apparent (pS.01) in concentrations of non-labeled Alac accumulated within explants

(figure 14). At each of the treatment x period levels Hol- stein explants contained from 75-150% more Alac than their

Angus counterparts (pS.01). Accumulation of Alac in explants also increased over time of incubation (p<.05). Thus concentrations of secreted Alac present in media varied in a manner similar to that of non-secreted.Alac detected in homogenates.

Synthesis versus Secretion of Proteins £Y Cultured Mammary Explants from Angus and Holstein Heifers

Total synthesis (media and homogenate quantities combined) of proteins, and percent of synthesized proteins ultimately secreted into medium by cultured mammary explants of beef and dairy heifers are illustrated in table 4. Hol-

stein explant cultures synthesized greater (p<.0005) total amounts of 3 H-TP combined over all treatments and periods, than did Angus cultures. In addition, approximately two- fold higher synthetic rates of TP were observed in Holstein 87

HOURS 96 iJ -23.3 41. 7 195. 7* 48 * 16. 3 123.9 30.9 47.8

TAT LEGEND: TRT -RB -RP (=:]HB C:=JHP

Figure 13: Secretion of non-labeled Alpha-lactalbumin into media by heifer explants exposed to 3 HH-amino acids during culture.

Units in pg/mg/ml/24 h. SEM • 13.8 *Greater (p=.0001) than corresponding Angus mean.

Note: values from one Holstein heifer were excluded from 96 h means due to exceptionally high ( 2000 pg) concentrations. 88

HOURS

290 * 100.5 2 41. 5*

** 148.4 8 0. 1

TRT LEGEND: TRT -AB -AP c::J HB c::J HP

Figure 14: Mean non-labeled alpha-lactalburnin in homogenates of heifer explants exposed to 3 H- arnino acids during culture.

Units in pg/mg/ml/24 h. SEM .• 18. 7 *Greater (p=.0001) than corresponding Angus mean. ** Greater (p~.01) than corresponding Angus mean.

Note: values from one Holstein heifer were excluded from 96 h means. 89

than in Angus cultures within each of the treatment x period

subgroups (pS.0001).

Elevated synthetic rates were apparently not accompanied by increased percent of TP secreted into medium. Secretory percentages were not different between P-treated Angus and

Holstein cultures at 48 h (p>.10), however a tendency for higher percent secretion by Angus explants was evident among

P-treated explants at this period (p<.10). Angus explants clearly secreted a greater percentage of newly-synthesized

TP at 96 h in both Band P-treated cultures, than similar

cultures of Holstein explants (pS.0001). Breed and period

effects on TP-secretory percent evidently interacted as well

(p<.05). Thus increased synthesis of TP in Holstein explants did not result in greater percent secretion of TP at

96 h. Indeed the opposite effect appeared to be true, that

is, increased synthesis in Holstein explants was accompanied by proportionally lesser secretion of 3 H-TP.

Total synthesis of 3 H-Alac was more than doubled (p<.005)

in Holstein cultures over that observed in Angus cultures in

each of the treatment-period groups (table 4). Breeds also

differed over all treatments and periods (p<.05). Thus to-

tal synthesis of 3 H-Alac followed a pattern similar to that

previously shown for 3 H-TP synthesis (table 4). Percent of

synthesized 3 H-Alac subsequently secreted into media was in- 90

TABLE 4

Synthesis and secretion of milk proteins by cultured mammary explants from Angus and Holstein heifers.

I I Time of Culture (h) I I 48 I I - ""°g6" I I BxT~ I I Total I Percent I I Total I Percent IParameterlGroupl I Synthesis I Secreted! I Synthesis I Secreted

AB I I 117.3 54. 3*** 11 112.4 71.2* HB 11 265. 7* 48.2 I I 274.1* 39.4 II 11 AP 11 129.3 53.8 I I 114.5 65. o* HP 11 258 .o• 51.1 11 249. 3* 44.4

AB 11 24.8 75.4- 11 33.3 I 87.7* HB 11 78. 3** 68.1 I I 76.8** I 61.4 II 11 I AP 11 27. 6 76.o I I 30.9 I 83 .o* HP 11 75_3*" 72.9 I I 00.1** I 68.1

AB 11 88. 0 21. 3 11 134.5 21. 8 HB 11 272. 3"' 41. 1* 11 491. 4* 36. 6** C Alac II 11 AP 11 110. 9 29.2 I I 142.2 28.1 HP I I 189. o*· 21.0 I I 437.2* 36.9**

*Greater (p<.0005) than corresponding mean of opposite breed. **Greater (p<.05) than corresponding mean of opposite breed. •••Greater (pS.10) than corresponding mean of opposite breed. ~Cpm/mg/ml; SEM for total synthesis= 17.6, for percent secreted= 2.3. bCpm/mg/ml; SEM for total synthesis= 10.8, for percent secreted= 1.5. cPg/mg/ml; SEM for total synthesis= 24.4, for percent secreted= 3.3. 4 BxT = Breed by Treatment. 91

creased in Angus compared to Holstein cultures following exposure to Bat 48 h (pS.0001), as well as in cultures exposed to B or Pat 96 h (pS.0001). However secretory percentages did not differ (p>.10) between breeds among cultures treated with Pat 48 h (table 4). Breed and period

(pS.01), as well as breed, treatment and period (p<.10), interacted to affect percent of newly synthesized 3 H-Alac secreted into media. Similar to synthesis and secretion of

3 H-TP, increased synthesis of Alac in Holstein cultures was not associated with increased secretion of product on a percentage basis.

Estimates of non-labeled Alac concentrations are not directly comparable to measures of 3 H-proteins, because non- labeled Alac may have been synthesized and/or secreted prior to the sampling period indicated. Thus, discrepancies in relative secretory patterns between non-labeled and

3 H-labeled Alac synthesis and secretion (table 4) may be accounted for by this circumstance. Absolute mean concentrations of Alac accumulated in media and homogenate were greater in Holstein than in Angus cultures treated with B or

Pat 48 h (pS.01) and 96 h (pS.0001). Percent secretion of

Alac was greater (pS.0001) in B-treated Holstein explants at

48 h than in comparable Angus explants, however no difference was detected between mean secretory percentages for P- 92

treated Holstein and Angus cultures during this period

(p>.10). In addition, higher percentages of Alac were secreted into media by Holstein explants cultured in B

(p<.005), and P (p<.05), at 96 h than those recorded for An- gus explants. Mean accumulation of Alac differed overall by breed (pS.01), as well as period (pS.05).

Combined secretory percent did not differ by breed

(p>.10), however it was apparently affected by a breed x treatment x period interaction (p<.10). The increased accumulation, accompanied by greater percent secretion of non- labeled Alac from Holstein explants exposed to Pat 96 his in contrast to observations of 3 H-Alac and 3H-TP synthetic and secretory patterns (table 4). As stated above, this in- consistency may be explained by the difference between 3 H- and non- 3 H-protein measurements. Tritium-labeled proteins must have been synthesized during the 24 h of incubation ending at the indicated time, whereas accumulated Alac

(non- 3 H) may have been synthesized at any previous time.

Correlations

Strong associations were observed between concentrations of 3 H-TP and 3 H-Alac detected in media from Angus (r=.95) and Holstein (r=.91) explant cultures. These parameters were also highly correlated in homogenates of explants, with 93

coefficients of .98 and .94 for Angus and Holstein tissue, respectively. Concentrations of Alac accumulated in media were associated with those detected within explant homogenates in Angus {r=.40) and Holstein (r=.85) cultures, as well. Thus 3 H-labeled TP and Alac measurements were in good agreement whether in media or homogenate samples. Concen- trations of non-labeled Alac in media were related to those in homogenates as well. Correlation coefficients for-other pairs of parameters are presented in Appendix d.

3 H-Thymidine Incorporation into Mammary Explant DNA

Incorporation of 3 H-thymidine into DNA of mammary explants subjected to short-term culture at zero time is sum- m~rized in table 5. Explants from Holstein heifers accumulated twice as much 3 H-thymidine (p<.10) as those from Angus heifers. Thus Holstein mammary explants were apparently more active in terms of cell division, as well as in production of proteins (see previous figures), than explants obtained from Angus heifers.

Serum Prolactin and Aloha-lactalbumin

Concentrations of prolactin in serum from Angus and Hol- stein heifers during the steroid pretreatment period are illustrated in table 6. Serum prolactin did not differ 94

TABLE 5

Incorporation of 3 H-thymidine into DNA of mammary explants from Angus and Holstein heifers.

I Breed I N !Mean CPM:\ S.E.M. I I I I 1-1 I Angus (A) I 3b I 80.8 32.7 I I I !Holstein (H) I 5b I 162.7* 25.3 I I I

3 Least squares mean (cpm/mg/h) 3 H-thymidine incorporation, ± standard error of mean.

bsome subjects lacked sufficient mammary tissue for this analysis after assignment of explants to culture.

:( Greater (p<.10) than mean for Angus. 95

TABLE 6

Concentrations of prolactin in serum of Angus and Holstein heifers during steroid pretreatment.

I I I Mean I Breed I Daysa I N 6 I Prolactirfl S.E.M. I I I I I 1-1 I Angus (A) I 1-7 (I) I 23 I 2.0 I .4 I 8-15 (PI)I 24 I 2.8 I . 7 I I I I Holstein (H) I 1-7 (I) I 28 I 22.Sx I 3.0 I 8-15 (PI) I 31 I 20.8 I 2.9 I I I I

QSamples collected during-, (I), or post-injection (PI) of estradiol and progesterone. b Samples were collected randomly throughout the pretreatment period. Not every subject was sampled on a given day.

cMean concentrations of serum prolactin (ng/ml), ± standard error of mean.

Greater (p<.0005) than corresponding mean for Angus. 96

(p>.10) between injection (days 1-7), and post-injection

(days 8-15) in samples from either breed. However Holstein heifers averaged 10-fold greater concentrations (p<.0005) of prolactin in serum over the 15 d of pretreatment than Angus heifers.

This difference may have resulted from environmental influences on prolactin secretion. Angus heifers were housed out-of-doors during October and November, whereas Holsteins were housed inside, under similar photoperiod. Although daylength was controlled for between the breeds, temperatures were certainly different and may account for the lower prolactin concentrations in Angus heifers.

Concentrations of Alac were also quantified in serum samples from heifers of both breeds, however concentrations were generally undetectable. Chapter V

DISCUSSION

EXPERIMENT I. MALES

General

The ability of mammary tissue from males of various species

to produce milk has been reported. Spontaneous lactation has been recorded in goats (Nair et al., 1981; Asdell et

al., 1936). In addition, experimental manipulation has re-

sulted in milk secretion from male cats (Turner and DeMoss,

1934), mice (Turner and Gomez,1934), and humans (Huggins and

Dao, 1954), as well as in cattle (Williams and Turner, 1961;

Kiddy et al., 1965). Furthermore, Freeman and Topper

(1978a,b) detected lactose synthetase in mammary tissue from male mice following steroid therapy. The present study supports these observations, that is, mammary tissue from males

of beef and dairy breeds is shown to be capable of de nova

synthesis and secretion of milk proteins (Alac and presump-

tive casein). Production of milk components by ruminant mammary tissue in response to hormonal stimuli in vitro has been commonly accepted as as index of lactogenesis (Collier et al., 1977; Skarda et al., 1979; Delouis et al., 1980;

Goodman et al., 1983), therefore it is reasonable to consid-

er the mammary tissue in these experiments as actively un- dergoing lactogenesis.

97 98

Breed Differences

The primary objectives of this experiment were to ascer- tain whether mammary tissue from prepubertal bulls is capable of producing measurable quantities of secretory product in response to hormonal stimuli, and if so, to determine if differences in genetic potential for milk production (beef breed vs dairy breed) would be reflected in these measurements. Thus development of a "bioassay" for genetic potential to produce milk was sought. The above objectives have been met in the present study.

The rationale for using the milk protein, Alac as an index of lactogenesis has been described by Goodman (1981).

Indeed synthesis and/or secretion of Alac has been utilized as a marker of mammary differentiation ·in bovine (Goodman et al., 1983) and ovine (Byatt, 1983) mammary tissue as well as in non-ruminant studies as reviewed by Forsyth (1983). The present study indicates that mammary tissue from bulls is also capable of synthesizing and secreting Alac, in agreement with the reports cited above. This ability to produce

Alac was common to both breeds, however the degree of production differed between the breeds. Mammary tissue from

Angus bulls was more active than Holstein tissue in terms of total synthesis of Alac (table 1), as well as in secretion of 3 H- (figure 3) and non- 3 H Alac (figures 1 and 6). Alt- 99

hough breeds did not differ overall within every parameter

(eg. table 1), statistically significant differences were apparent between ·breed means for at least two treatment x period levels in these cases. The exception to greater productivity by Angus tissue was in concentrations of 3 H-Alac detected in homogenized explants (figure 5), which were greater in Holstein cultures at each of the treatment x period levels. This observation is likely explained by more efficient secretion of newly synthesized Alac in Angus tissue. Examination of figures 3 and 5 demonstrates that the portion of 3 H-Alac retained in explants was but a small fraction of the total synthesized during each treatment-period, particularly in the case of Angus. Results presented in table 1 lend further support to this argument, emphasiz- ing the significantly greater percent secretion of 3 H-Alac in Angus cultures. Furthermore, figure 7 shows that concentrations of Alac detected by RIA were greater in homogenates of Angus explants. Therefore figure 5 probably indicates a retardation in onset of secretion in Holstein relative to

Angus tissue, rather than greater synthesis of 3 H-Alac by

Holstein explants. Thus, Angus tissue was superior to that from Holsteins in both synthesis and secretion of 3 H-Alac.

Further parameters examined include synthesis and secretion of 3 H-total protein (TP; acid precipitable protein 100

fraction). It is important to note that TP is a non-specific measurement (as far as lactogenesis is concerned), particularly when determined in tissue homogenates. This is because all intracellular proteins would be precipitated, as opposed to milk-specific proteins alone. However increased

3 H-TP in homogenates of mammary tissue is likely to reflect syntheses of enzymes and proteins required for milk synthesis and secretion in addition to unsecreted milk proteins.

Therefore this parameter may be a valid indicator of lactogenesis. In fact, Skarda et al. (1982) measured synthesis of TCA precipitable proteins as an indicator of mammary differentiation and found that it agreed well with syntheses of lipid and casein in goat mammary explants cultured in medium containing insulin, cortisol and prolactin. Concentrations of 3 H-TP detected in media are representative of secretory protein released from cells into media (provided no cell ly- sis has occurred). It is reasonable to expect that this protein fraction would be largely comprised of casein, since caseins make up 82% of total protein in cow's milk (Davies et al., 1983). In addition, Alac content was determined in samples and could be subtracted from TP to yield a functional estimate of casein concentrations. However as reported in the results, the portion of 3 H-TP made up of 3 H-Alac ranged from 30-60%. Clearly then, the remaining protein, 101 even if entirely casein, would not reach the percentage ex pected. This may be due to selective stimulation of Alac synthesis by synergism of prolactin with T 3 , as described by Vonderhaar (1977) in mice. Terada and Oka (1982) showed that this synergism is indeed Alac specific, since casein and casein-mRNA syntheses were not affected. Ono et al. (1981) described differential regulation of Alac and casein syntheses in mouse mammary glands, depending on relative concentrations of corticoid added to media containing insu lin and prolactin. Similar results have been reported in ruminants by Skarda et al. (1982) who found that addition of T 3 did not augment synthesis of casein or TP above levels attributed to prolactin stimulation alone. Likewise, Nar dacci et al. (1978) found that Alac and casein mRNAs were differentially induced in murine mammary tissue. Thus the relatively lower percent of TP made up by casein in this study is not without precedent, and probably indicates spe cific stimulation of Alac synthesis by prolactin-T 3 syner gism. Precedents for using casein synthesis as an index of mammary cell activity in ruminants have been established by Gertler et al. (1982) and Skarda et al. (1978, 1982). 3 Synthesis and secretion of H-TP followed the pattern ob served above for Alac. Specifically, Angus bull tissue syn thesized (table 1), and secreted (figure 2), significantly 102 greater quantities of TP than Holstein tissue at each of the treatment x period levels as well as overall. Similar to 3 3 H-Alac, H-TP was significantly elevated in Holstein homo genates compared with those from Angus mammary explant homo genates (figure 4). As above, this phenomenon is probably attributable to lesser development of secretory mechanisms and thus greater retention of protein in Holstein tissue. If this reasoning holds true then the reduced concentrations 3 of H-TP in Angus tissue is a further indication of greater differentiation, specifically of secretory mechanisms, com pared to Holstein tissue. These data are indicative of the potential for develop ment of a bioassay for genetic potential to secrete milk. Although there is obviously no direct evidence to link the parameters measured in the present study with a bull's sub sequent performance as a sire, it is logical that the abili ty to differentiate animals from high- (dairy), and low (beef) milk producing breeds might be applicable to select ing genetically superior bulls from within a breed. Indeed, in every analysis variation between individual animals with in breeds was statistically significant. Thus the possibil ity of ranking individual bulls existed. The idea of secre tory proteins reflecting an animal's genetic makeup has been utilized by Kiddy et al. (1965) in tracing heritability of 103

variant forms of the milk protein beta-lactoglobulin in mammary secretion from bulls as well as cows. Similarly, Gert- ler et al. (1982) have reported tremendous differences in casein synthesis by mammary tissues from over 40 lactating cows. These workers suggested that this variation may be attributed to differing secretory capabilities of individual animals which could be ultimately related to genetic differences.

The fact that explants from Angus bulls were more active in synthesis and secretion of proteins suggests that less responsive animals (Holsteins) would be superior sires, if the assumption that Holsteins possess better genetics for milk production holds true. This option is viable for selection, however it is physiologically problematic. Possi- ble explanations for this paradox will be considered below

(see Other Considerations).

Effect of Treatment and Incubation Time on Productive Indices-

As stated previously, the primary concern of this study involved differentiating breeds based on the parameters quantified. For this reason, breed means were compared for each treatment x period level using orthogonal contrasts. Howev- er overall effects of treatment and period (and interactions) were included in the statistical model (appendix b) 104

and can be reasonably discussed in the context of this study.

Presence of prolactin in culture medium had a generally

stimulatory effect on synthesis and secretion of proteins.

Similar to the findings of Goodman et al. (1983) the data

indicate a positive effect of prolactin on synthesis and

secretion of Alac as evidenced by greater concentrations of

newly synthesized Alac in homogenates (figure 5), as well as

increased amounts of non-labeled Alac in media and homogen-

ates (figures 1,6,7). The ability of prolactin to enhance

total synthesis and percent secretion of 3 H-Alac became more

apparent in the later time period (table 1). This "lag

time" before prolactin effects are maximal is in agreement

with the results of Goodman et al. (1983).

Synthesis of 3 H-TP (and therefore casein) by bull mammary

tissue tended to be greater (p<.10) in the presence of pro-

lactin (figure 5). As shown in table 1, prolactin also sti-

mulated cumulative synthesis and secretory percent of 3 H-TP

although these effects were not evident until the 96 h per-

iod (treatment x period, p<.05). These data indicate that

like Alac, synthesis and secretion of TP (casein) were en-

hanced by addition of prolactin to culture medium. Such

findings are in agreement with reports by Skarda et al.

(1982a;b) in goats, as well as those of Gertler et al. 105

(1982) in lactating bovine mammary tissue. It seems clear then, that mammary tissue from prepubertal bulls is comparable to that from ruminant females, in characteristics of response to in vitro culture in the presence of lactogenic hormones.

Synthesis versus Secretion of Milk Proteins

The question of whether synthesis and secretion of milk products occur as synchronized events has been raised by

Cowie et al. (1980). Indeed evidence for dissociation (or independent control) of these processes has been cited in mice (Chatterton et al., 1975), as well as rabbits and ewes

(Ollivier-Bousquet and Denamur, 1975). As the present study yielded information on de novo synthesis and subsequent secretion of milk proteins (table 1), the topic is germane to this discussion. Heald and Saacke, (1972) reported that incorporation of 3 H-leucine into mammary secretory proteins proceeded similarly whether in vivo, or in vitro, in mammary tissue from mice. Autoradiographic techniques were utilized to observe the uptake of 3 H-label from blood or media and subsequent passage through the cell and discharge into the lumen in the form of 3 H-protein. However Forsyth (1983) has described mammary explants as closed systems, with large retention of milk proteins and thus relatively little secre- 106

tion due to closure of ducts. The present findings contrad- ict the observations of Forsyth (1983) in that newly synthesized proteins (Alac and TP) were effectively secreted into media. As illustrated in table l, greater than 80% of

3 H-proteins were recovered from media (with the remainder in homogenates) regardless of breed, treatment or period.

These data are consistent with those of Goodman et al.

(1983) who recovered over 90% of 3 H-leucine labeled Alac from media as opposed to homogenates, of bovine mammary explants cultured in the presence of prolactin.

Differences in secretory activity between breeds are apparent from the data in table 1. Angus bull explants not only synthesized, but also secretea greater quantities of

3 H-TP and 3 H-Alac into media. This difference probably represents a greater degree of cellular differentiation, en- compassing both synthetic and secretory mechanisms, in Angus over Holstein tissue. The possibility of intrinsic differential in secretory ability of differing breeds is rendered unlikely by the similarity of secretory percentages for non-labeled Alac across both breeds, in spite of greater protein production by Angus explants (table 1). Apparently newly synthesized proteins are secreted in preference to preexisting molecules, perhaps as part of a coupled process.

Such a coupling of synthetic and secretory processes can be 107

inferred from the data of Heald and Saacke, (1972) in which

3 H-leucine traveled as a pulse through protein-synthetic and secretory machinery, and was released into alveolar lumina within one hour of exposure to isotope. Perhaps coupling of synthesis and secretion merely represents differentiation of independently regulated mechanisms occurring simultaneously.

It seems logical however that these mechanisms would be subject to common regulation at some level, since either synthesis without secretion, or activation of secretory mechanisms in the absence of product would be biologically uneconomical for the cell.

Other Considerations

Study of lactogenesis through organ culture of mammary gland explants has been a powerful and widely accepted research tool. However Forsyth (1971) has cautioned that such experiments should include some measure of tissue viability in vitro, (generally histological examination), to allow for proper interpretation of results. In the present study, incorporation of 3 H-amino acids into secretory protein by mammary explants provides a functional index of explant viability. As portrayed in table 1, mammary explants remained active in the synthesis of Alac and TP through 96 h of incubation. Overall period means were different (p=.06) for 108

Alac synthesis, however overall synthesis actually increased over time, due mainly to the appearance of prolactin stimulation at the later period {pS.0001). These data indicate that explants remained viable throughout the incubation period, and indeed became more active over time, at least in the presence of prolactin.

Correlation analyses in the present study have been useful in determining the relationships between various parameters. Large positive correlations between 3 H-TP and 3 H-Alac concentrations were evident in media and homogenates from both breeds. The findings indicate that these two parameters are both reliable indicators of protein synthesis and secretion by the explants. Thus similar inferences could be drawn from either measurement. This is not always the case, as studies by Ono et al. (1981) and Terada and Oka, (1982) have demonstrated differential expression of casein and Alac syntheses in mouse mammary explants. Apparently the hormonal combination utilized herein was capable of inducing differentiation of mechanisms for synthesis and secretion of both Alac and casein (TP).

Perhaps the most intriguing aspect of this experiment was the greater degree of response in Angus over Holstein bull mammary tissue. This finding was in contrast to the initial hypothesis which predicted that Holstein bull explants, 109

possessing superior genetics for milk production, should express this superiority through increased production of milk proteins. Possible explanations for the relatively greater response of Angus tissue are discussed below.

The most probable cause of greater productivity by Angus tissue lies not in true expression of superior genetic potential but rather in response to steroid pretreatment (induced lactation scheme). Gross observation of mammary glands at the time of explantation suggested a greater degree of development in Angus over Holstein glands. Angus bull glands showed evidence of proliferation of mammary epithelium into surrounding fat pad and stroma. Ductular lumina appeared larger and ducts were more widely branched and ended in presumptive terminal end buds more frequently than in Holstein tissue. Total area occupied by parenchyrna also appeared greater in Angus glands. Thus subjective observations of gross mammary gland anatomy suggested an elevated response to steroid therapy by Angus compared with Holstein bulls. It is reasonable to expect that animals more responsive to pretreatment should subsequently be more active in vitro. Indeed Turner et al. (1956) attributed differential success in induced lactations to variable sensitivity of individual animals to steroid therapy. 110

Unfortunately data from parameters intended to quantify response to pretreatment were not supportive of the qualita- tive assessments. Serum prolactin concentrations during the induction period were significantly greater in Holstein versus Angus bulls (table 3). Although Holstein bulls were generally placed on experiment earlier in the year than An- gus bulls (9/6-10/31 vs 10/28-11/7), considerable overlap between groups existed, and 10 of the 12 subjects were slaughtered within one month. Thus effects of daylength and temperature were unlikely to have caused the observed breed difference in serum prolactin concentrations. Ohlson et al.

(1981) reported increased serum concentrations of prolactin and growth hormone in bull calves of a larger, faster grow- ing beef breed (Simmental), than in a smaller, slower grow- ing breed (Hereford). Furthermore, increased prolactin concentrations during induction of lactation in cattle have been positively correlated with milk yields (Erb et al.,

1976; Mollett et al., 1976). Thus circulating concentrations of hormones may be related to productive function. However a direct relationship between prolactin concentrations and production of proteins was not evident in the present study.

Rates of incorporation of 3 H-thymidine into DNA at zero time were approximately 30% greater in Holstein compared 111

with Angus mammary tissue, although breed means were not

statistically different (p>.10; table 2). Therefore differ-

ences between breeds in synthetic and secretory activity as

well as in apparent degree of glandular development were not

the result of differential cell division, at least that oc-

curring at zero time.

The lack of agreement between indices of response to

steroid pretreatment (serum prolactin and 3 H-thyrnidine in-

corporation) and gross observations of mammary gland devel-

opment emphasize the complex timing of induction of lacta-

tion. The greater response of Angus bull tissue to hormonal

stimuli in vitro may be explained by increased development

following steroid therapy, however reasons for Angus exhi-

biting greater sensitivity to estrogen-progesterone treat-

ment are unknown. Perhaps had the pretreatment period been

extended from 14 to 21 days similar to the method of Howe et

al. (1975) development of Holstein glands may have equalled

or surpassed that of Angus glands. When developmental

events which normally occur over 280 d of gestation are com-

pressed into a 14-d period, (as in induction of lactation in

females), day-to-day changes must be tremendous. Thus, even

a small difference in response time to steroid induction

might be translated into a dramatic difference in response

in vitro. This reasoning is purely speculative and underly- 112

ing factors remain unknown. Clearly, it would be of inter est to conduct a similar study, omitting the steroid pre treatment. Although the amount of parenchyma available for explantation would be reduced, in vitro results would be re flective of tissue response to culture conditions, without the complicating factor of response to steroid therapy. Such research could potentially answer many questions, prin cipally why mammary explants from Angus bulls were more res ponsive than those from Holsteins; and could further define optimal conditions for a bioassay for genetic potential for milk production.

EXPERIMENT II. FEMALES General The ultimate goal of the present study was to determine whether differences in genetic potential for milk production among individual animals could be exposed through mammary tissue responses to hormonal stimuli. As discussed previ ously, such research could lead to applications in animal industry, particularly in sire selection. Thus, bulls of high-, and low-milk-producing breeds were obtained as sub jects. However, due to the relatively unknown nature of mammary gland response to hormones in the male, the second phase of the study included females of the same breeds. In 113

this manner the females were expected to serve as a positive control. Furthermore, study of responses by prepubertal heifer mammary gland seemed pertinent to gaining understanding of breed differences among females, as well as providing insights on the relative ability of young animals to respond to hormone treatment.

Breed Differences

In contrast to the previous experiment involving bulls, responses of heifer mammary tissue to lactogenic stimuli were in accord with the initial hypothesis. Explants of

Holstein heifer mammary gland were more active in synthesis and secretion of milk proteins than were those from Angus heifers. This assertion held true in terms of absolute concentrations of Alac and TP synthesized and secreted from cultured explants, (figures 8-14; table 4), although not in relative percent secretion of newly synthesized proteins

(table 4). Thus productive responses in vitro reflected the difference in milk production normally ascribed to these breeds.

The concept of comparing animals from breeds demonstrated to have differing productive potential is not novel. Previ- ous studies have contrasted circulating hormone profiles in high- and low-milk-producing breeds of cattle (Falconer et 114

al., 1980; Hart et al., 1980), different beef breeds (Keller et al., 1979), and sheep breeds with differing ovulation rates (Wheeler et al., 1977). Recent work by Davis et al.

(1983) compared milk yields and udder volumes of Jersey cows and heifers from high- and low-breeding-index groups. Re- sults of these studies have generally indicated differences between breeds or groups reflecting differences in their genetic complement. Thus, previous studies are supportive of the present findings.

Effect of Treatment on Protein Synthesis and Secretion

The profound stimulatory effect of prolactin on in vitro synthesis and secretion of milk proteins by prepartum (Good- man et al., 1983), and lactating (Gertler et al., 1982), bovine mammary tissue has been demonstrated. Thus, the relative lack of prolactin-responsiveness by explants from heifers in the present study is surprising. Some indication of prolactin effect may be derived from the data shown in figure 8. Although no overall effect of prolactin is evident, Holstein heifer explants clearly secreted more Alac into media in the presence of prolactin at 48 and 72 h than explants from other groups at comparable times. This instance however, was the sole example of tissue response to prolactin, as no effect was detected in any of the remaining 115

parameters. Several possible explanations for these results can be considered.

That the mammary tissue utilized in this experiment was derived from glands of prepubertal heifers may be an important factor. Skarda et al. (1978) have reported that in vitro lactogenic responses to prolactin of goat mammary tissue vary dramatically with stage of ontogenesis of the donor.

Explants from 2- or 24-day-old goats, as well as those bey- ond 8 wk of pregnancy, demonstrated positive responses to prolactin. However, tissue from goats (virgins and pregnant does) between these stages did not exhibit lactogenic responses to prolactin, even when duration of culture and concentrations of hormone were increased. The heifers used in the present study were at an age (6-8 mo) corresponding to the inactive period in goats, thus it it possible that the mammary explants from these donors remained refractory to prolactin, despite steroid priming in vivo.

Carryover effects of prolactin bound to membrane receptors in vivo, on subsequent responses in vitro represent another possible explanation for lack of response-to this hormone. Such carryover effects have been described for glucocorticoids on responses of mouse mammary tissue in vitro (Bolander et al., 1979). However the 10-fold increase in serum prolactin concentrations in Holstein over Angus 116 heifers (table 6) contradicts this thesis. If retention of prolactin in explants was responsible for blocking the ap pearance of hormonal effects in vitro, one might expect that Angus explants (exposed to far less prolactin in vivo) would exhibit relatively greater responses to prolactin in vitro. This was apparently not the case. The possibility that se rum concentrations of prolactin in Angus heifers were suffi cient to effect blockage of effects in vitro remains, howev er, data from the previous experiment (see Experiment I: Males) is in contrast to such a supposition. A final point to consider on the inability of these ex plants to display lactogenic responses to prolactin has been forwarded by Gertler et al. (1983). These workers suggested that secretion of milk proteins in response to hormonal sti muli in vitro is masked by leakage of proteins formed in vivo. Masking of hormonal effects in this manner apparently continued until 72 h in culture (following three changes of medium). Goodman, (1981) also alluded to similar "dumping" effects, although these were comparatively short-lived (ap proximately 8 h). This explanation is initially attractive since high concentrations of Alac in medium from Holstein cultures at 24 h (figure 8) suggest just such a dumping ef fect. However analysis of synthesis and secretion of 3 H-proteins, which could not have been influenced by prior 117 synthesis, failed to indicate a positive effect of prolac tin. Thus, proposed masking of prolactin effects in vitro does not provide a satisfactory explanation for the data ob served. The lack of prolactin stimulation of protein synthesis and secretion in the present experiment remains enigmatic, particularly when compared to the positive res ponses observed in males of similar age and breeds (see Ex periment I: Males). It is difficult to construct a reason able postulate to account for these results; perhaps the only plausible explanation lies in unknown developmental processes which result in tissue that is unresponsive to prolactin induction of protein synthesis and secretion, at this stage of ontogenesis.

Synthesis versus Secretion of Milk Proteins Synthetic and secretory mechanisms in mammary epithelial cells are apparently under independent regulation (Chatter ton et al., 1975; Ollivier-Bousquet and Denamur, 1975; Cowie et al., 1980). Nonetheless, normal parturient activation of the mammary gland requires differentiation of both of these pathways to allow for optimal milk production. It would seem then, that non-coupled differentiation of these systems represents an abnormal (or at least suboptimal) state. A comparison of relative secretory activity with protein syn- 118 thetic activity (table 4) suggests that among heifers in the present study, synthesis and secretion were not coupled events. Holstein heifer explants synthesized significantly 3 3 greater quantities of H-TP and H-Alac at each treatment x period level, whereas secretory percentages were signifi cantly elevated in Angus explants for three of the four treatment x periods. It may be argued that absolute concentrations of protein secreted were greater among Holstein ex plants, which is indeed the case. It seems logical however, that the proportionally lower percent secretion in more syn thetically active cells may represent asynchronous develop ment of these two mechanisms. This concept is supported by 3 the increase over time in concentrations of H-TP and 3 H-Alac in homogenates from both breeds (figures 11 and 12), without concurrent increases in quantities of these proteins in medium (figures 9 and 10). Perhaps prolactin-responsive ness is particularly necessary for differentiation of secre tory function. Examination of concentrations of non-labeled Alac accumulated in medium, and percent secretion of this fraction (table 4) indicates that Holstein tissue accumulat ed and secreted greater concentrations of non-radiolabeled Alac than Angus tissue. These data imply that lower secre 3 tory percentages for H-proteins in Holstein cultures may reflect competition for secretory packaging and transport, 119

by non-labeled proteins. Such competition would be in contrast with the preferential secretion of newly formed proteins (see Experiment I) proposed for explants of bull mammary gland.

Despite the apparent difference between males and females in synchrony of synthetic and secretory activity, these re-

sults are compatible with the concept of differential regu-

lation of the two processes (Cowie et al., 1980). Whereas male tissues apparently developed these mechanisms in synchrony, female tissue may have lacked the ability to fully

respond to prolactin stimulation, possibly because of inhe-

rent inactivity at this stage of development as proposed by

Skarda et al. (1978). However the underlying mechanisms which differentially control these processes remain unclear.

Other Considerations

Milk yield is determined by the number of functional secretory cells in the mammary gland and the relative produc-

tion of each cell (Mepham, 1983). This relationship can be

applied to production of milk proteins by mammary explants

in vitro, as well. Therefore the elevated production of proteins by explants from Holstein heifers may be attributed

to greater cell numbers and/or increased cell activity.

Although absolute numbers of secretory cells per explant 120

were not quantified, rates of incorporation of 3 H-thymidine into DNA were doubled in Holstein over Angus explants

(p<.10; table 5), prior to initiation of culture. If this elevated rate of cell division is indicative of that during the pretreatment period, then cell numbers per explant may have likewise been greater in Holstein than in Angus explants. Gross observations of mammary tissue during explantation tended to support this idea. Holstein glands appeared to contain greater proportions of parenchyma:stroma, and presumptive parenchyma had invaded the mammary fat pad to a greater extent than in Angus glands. On the other hand, serum concentrations of prolactin during pretreatment were increased ten-fold in Holstein compared with Angus heifers (p<.0005; table 6). Plasma prolactin concentrations have been related to success of induced lactation, (Erb et al., 1978; Mollett et al., 1978), presumably through stimulation of secretory cell activity. Akers et al. (1981) have shown conclusively that prolactin is essential for terminal differentiation of mammary secretory cells and optimal milk production. Thus increased levels of circulating prolactin may have induced greater differentiation in Holstein tissue.

It would appear from the above discussion that cell numbers and cell differentiation may have been enhanced by steroid pretreatment to a greater degree in Holstein than in 121

Angus heifers. Perhaps the greater responsiveness of Hol- stein heifer tissue to hormonal stimuli represents a funda- mental genetic difference between these breeds. Presence of prolactin in media did not result in positive lactogenic responses. However the initial advantage in differentiation enjoyed by Holsteins, as evidenced by increased production of proteins, was maintained throughout the culture period.

If greater mammary gland sensitivity to hormones is the actual mechanism which confers an enhanced ability to produce milk upon Holstein compared to Angus females, study of breed differences may be focussed upon two points. First, are high-milk producing animals inherently more sensitive to hormonal stimuli, perhaps due to increased numbers of lactogenic hormone receptors on cell membranes? Or do high-pro- ducers harbor an endocrine system which supplies a more optimal hormonal milieu for promoting glandular growth and differentiation? The answers to these questions may well define the difference between high- and low-producing breeds, as well as individuals within a breed. Such understanding would point the way for optimization of conditions conducive to greater milk production in dairy cattle. Chapter VI

SUMMARY AND CONCLUSIONS

The ability of mammary tissue from prepubertal bulls and heifers of beef and dairy breeds to respond to lactogenic hormonal stimuli through synthesis and secretion of milk proteins was studied. Experimental animals were 6- to 8- month-old Angus and Holstein cattle. All subjects were placed on an induced lactation regime (injection of estradiol and progesterone sc. for seven days; slaughter on day

15 after initial injection) to promote mammary growth. Mam- mary tissue was explanted, randomly assigned to culture dishes, and incubated for up to 96 h. Culture medium con- sisted of medium 199 supplemented with insulin, hydrocortisone and triiodothyronine (5 ug/ml, .5 ug/ml, and .65 ng/ml, respectively), as basal (B); or the above combination with the further addition of bovine prolactin (1 ug/ml) as stimulatory (P), medium. Selected dishes received B or P medium containing 3 H-amino acids ( 4uCi/ml final concentration), and explants were incubated in the presence of isotope for

24 h. Specific parameters quantified included concentrations of non-labeled alpha-lactalbumin (Alac), as well as

3 H-Alac and 3 H-total protein (TP) secreted into medium, and present in homogenates of explants.

122 123

Among cultures of bull mammary tissue, Angus explants secreted greater overall quantities of 3 H-TP (pS.01) and

3 H-Alac (p<.05) than Holstein explants. Secretion of non- labeled Alac was also increased in Angus cultures exposed to

Pat 48 and 72 h, compared with Holstein cultures at similar treatment periods (p<.01). Concentrations of proteins in homogenates behaved similarly, as 3 H-TP, 3 H-Alac and non-labeled Alac values were greater (pS.05) in Angus than in corresponding cultures of Holstein tissue for at least three of the four treatment periods. Overall synthesis and percent of total secreted by Angus tissue were greater than that by

Holsteins in every case for 3 H-TP and 3 H-Alac (pS.05).

Thus, the parameters quantified indicated a significant difference between breeds in production of milk proteins. This difference allowed for distinguishing between high- and low-producing breeds. In addition, production among individual animals varied significantly (p<.0001) thus subjects could have been ranked in order, by their protein-productive activity in vitro. Based on these findings, relative genetic potential for milk production of each bull could be de- duced.

Ability to respond to presence of prolactin in medium was also apparent in tissue from both breeds of bulls. Signifi- cant overall effects of prolactin on secretion of unlabeled 124

Alac (p<. 005), as well as on accumulati.on of 3 H-TP {p<. 05),

3 H-Alac (p<.10), and unlabeled Alac (p<.05) in explants were observed. Prolactin also increased total synthesis and percent secretion of 3 H-TP and 3 H-Alac in a time dependent manner (p<.05). I conclude that prolactin supplied the stimu-

lus to enhance synthesis and secretion of proteins by mammary explants from immature bulls.

In Experiment II, explants from Holstein heifers displayed greater protein-synthetic activity in vitro than those from Angus heifers. Concentrations of Alac secreted

into medium were elevated in Holstein compared to Angus cultures in three of eight treatment periods (p<.0005). Ove-

rall quantities of 3 H-TP and 3 H-Alac were greater in Hol-

stein culture media (p<.10) and homogenates (p<.01), as were

concentrations of unlabeled Alac in homogenized explants

(pS.01). Total syntheses of 3 H-TP and 3 H-Alac were likewise

greater in Holstein than Angus heifer explants (p<.05).

However secretion of newly synthesized proteins reached higher percentages in Angus explants, an effect that was ac- centuated over time (p<.05) for both 3 H-TP and 3 H-Alac.

These findings demonstrate that Holstein heifer mammary tis-

sue possessed a greater capacity to respond to lactogenic

stimuli, in vitro, compared to tissue from Angus females.

This result was expected in light of the inherent differen- 125

tial in milk production between these breeds. Similar to the bulls in the first experiment, individual heifers within a breed varied significantly, thus heifers too could be ranked based on protein production in vitro, presumably in- dicating relative genetic merit for milk production.

Mammary explants from heifers of both breeds were generally unresponsive to presence of prolactin in medium. No overall effect of treatment on any productive parameter was detected (p>.10). However Angus explants accumulated more

3 H-TP and 3 H-Alac in the presence of prolactin, while P- treated Holstein explants contained slightly less protein than those exposed to B (breed x treatment, p<.01). This relative lack of tissue response to prolactin is attributed to explants being in an unresponsive state due to effects of steroid pretreatment in vivo, and/or stage of ontogenesis.

I conclude that mammary tissue from prepubertal male and female cattle survives in culture and is capable of de novo synthesis and secretion of constituent proteins of milk, following hormonal stimulation in vivo and in vitro. Ana- lyses of synthetic and secretory activity allowed for sepa- ration of breeds (and individuals) based on relative genetic potential for milk production. Thus, these techniques, once optimized, show promise for applications in the sire selection industry. LITERATURE CITED

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APPENDICES

Appendix a

Immunoprecipitation

!Total counts added !Counts recovered 1% Recovery I I I 238,917 I 169,211 70.8 117,915 I 87,222 74.0 63,681 I 46,115 72.4 24,270 I 17,201 70.9 12,802 I 8,761 68.4 8,421 I 5,749 68.3 4,415 I 3,024 68.5 2,340 I 1,637 69.9 I TCA precipitation !Total counts added !Counts recovered 1% Recovery I I I 256,127 227,141 88.7 129,083 113,582 88.0 63,981 60,194 94.1 25,367 23,221 91. 5 13,792 11,823 85.7 8,994 8,080 89.8 4,669 4,315 92.4 2,620 2,336 89.2

135 136

Appendix b

Th• followinq table the analy••• of variance •••ociated with bull• utilized in the atudy.

MAI.AC BAI.AC MPTP MPALAC BPTP HPALAC AI.AC source• .df m m BS m m m

Breed (B) 1 114.61 87.81 1528.41 14.7 46.0 116.62 89.2

Animal(B) (A(B)) 10 33.81 21.94 165.74 14.94 17.94 22.44 148.84

Treatment (T) l 110.11 34.12 12.9 10.22 4.11 .6 778.41

BX T l 55.1 1.2 6.5 2.2 1.3 1.3 63.2 A(B) x T 10 24.6 5.91 50.92 1.41 1.02 19.54 56.82 Period (P) l 141.91 1.6 194.l 2.6 1.2 49.32 34.8 B x p l 2.1 42.9 112.6 3.1 .8 10.6 97.l

A(B) x P 10 15.8 19.44 105.44 3.74 2.64 10.11 70.71 T x p l 9.2 16.32 250.01 1.91 .4 e1.6• 25.2

BX TX P 1 24.4 11.01 9.1. 1.3 ., 3.1 36.6 B11ism1l 1L1. u ll..l _j L.6 ll,.Q

!'or Alac1 P, BxP, TxP, BxT:xP, df • 3; A(B)xP, Reaidual, df • 30. teated bJ A(B); T, B:xT teated by A(B)xT; P, BxP teated by A(B)xP. ++its ( x 10) for all parameter• except BPALAC I MS ( x 101 ). •~.10; z~.05; ~.01; 4 p<.000l aalac ••• Alac (RIA) from ..dia balac ••• Alac (RIA) from homogenate• aptp .••• 1B-Total protein precipitated froa aedia apalac •• 1B-Alac precipitated from aedia bptp •••• 1B-Total protein precipitated from b0m09enatea bpalac •• 1B-Alac precipitated from homogenate• alac •••• Alac (RIA) from ••di• of non-1B-culture• 137

Appendix b (cont.)

The following table depicts the analyses of variance associated with heifers utilized in the present study.

MAI.AC BALAC MPTP MPALAC BPTP BPALAC AI.AC

Smu::s:1+ !!S!! BS BS BS BS BS BS Breed (B) l 302.11 303.31 85.61 37.21 168.o• 205.6• 9965.8 Animal(B) (A(B)] 10 40.21 31.14 25.74 9.34 14.44 15.64 4212.34 Treatment (T) l 3.5 4.9 .l .0 .l .6 447.9 BX T l 19.l 4.1 .0 .l 3.2• 4.S• 389.7 A(B) X T 10 8.24 6.11 6.61 s.1• .3 .2 213.2 1 Period (P) l 85.7 131.91 .9 .3 .0 .2 5764.61

B X P 1 62.4 47.9 8.7 .s 13.7• 17.21 5678.51 A(B) X P 10 27.34 21.64 9.9• s.2• _91 1.01 1773.04 TxP 1 8.01 5.3 .9 .o .0 .6 45.8 BxTxP 1 6.41 .7 .4 .3 .8 .1 65.4 B111sm1l l...§. L.2 L.! .! ..6 il...B For Alac: P, BxP, TxP, BxTxP, df • 3; A(B)xP, Residual, df • 30. +a teated bJ A(B); T, BxT tested by A(B)xT; P, BxP tested by A(B)xP. ttMS ( x 10) for all parameters except BPALAC I MS ( x 101 ). 1 pS.10J 1 pS.05; 1 pS.01; 4 p<.0001 aalac .•• Alac (RIA) from halac ••. Alac (RIA) fr0111 homogenates aptp •••• 1B-Total protein precipitated froa media •• 1B-Alac precipitated from ..di• hptp •••• •s-Total protein precipitated from hoaogenatea hpalac •• •B-Alac precipitated from homogenate• alac •••• Alac (RIA) from media of non-1B-culture• 138

Appendix c

Correlation coefficients for protein parameters from cultured bull mammary tissue.

rnptp mpalac hptp hpalac malac halac

mptp Al I .91 4 .37 3 1 .37 3 .503 .302 HI I .86 4 .04 -.00 .282 .483 I I mpalac Al I .23 .28 .57 4 .24 1 HI I -.19 -.20 .18 .443 I I hptp Al I .503 .443 .393 HI I .95 4 .OS .15 I I hpalac Al I .21 .59 4 HI I .04 -.01 I I malac Al I .02 HI I .363 I i A denotes Angus; H denotes Holstein. mptp .... 3H-Total protein precipitated from media. mpalac .. 3H-Alac precipitated from media. hptp .... 3H-Total protein precipitated from homogenate. hpalac .. 3H-Alac precipitated from homogenate. malac ... Alac (RIA) from media. halac ... Alac (RIA) from homogenate. 1 pS.10; 2 pS.05; 3pS.01; 4 p<.0001 for Ho: lrl =O. 139

Appendix d

Correlation coefficients for protein parameters from cultured heifer mammary tissue.

mptp mpalac hptp hpalac malac halac mptp Al .95 4 I .14 .19 .18 .49 3 HI .91 4 1-.644 -.67 4 -.01 .12 I I mpalac Al 1-.11 -.07 .27 1 .56 4 HI 1-.604 -.61 4 .04 .19 I I hptp Al I .98 4 -.20 -.18 HI I .944 .01 .08 I I hpalac Al I - . 24 1 -.12 HI I -.02 .04 I I malac Al I .40 3 HI I .85 4 I I A denotes Angus; H denotes Holstein. mptp .... 3 H-Total protein precipitated from media. mpalac .. 3 H-Alac precipitated from media. hptp .... 3 H-Total protein precipitated from homogenate. hpalac .. 3 H-Alac precipitated from homogenate. malac ... Alac (RIA) from media. halac ... Alac (RIA) from homogenate. 1 pS.10; 2 pS.05; 3 pS.01; 4 p<.0001 for Ho: lrl =0. The vita has been removed from the scanned document