“ I 7 SENEGAL EVALUATION OF THE BOVINE ENDOMETRIUH TENN 5M flu Dara of M. 5. MECHIGM STATE UNIVERSITY Lee W Edwards Jr. 3971 ‘ THES'S -._..--- LIBRARY ”* Michigan Swazi: Urmwsjcy OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records 2‘4. - fl‘l\\\\ L' P94 .' “new” ‘ 3! «" \.‘..’¢"- 1‘ “Qfigkfififiw“' .:\tu ABSTRACT A HISTOCHEMICAL EVALUATION OF THE BOVINE ENDOMETRIUM By Lee Ellis Edwards Jr. The objective of this study was to histochemically monitor 2 enzymes in the bovine endometrium. The enzymes selected, glycogen phosphorylase and alkaline phosphatase,‘are sensitive to variations in levels of estro- gen and progesterone, and their histochemical activities reflect the effects of these hormones upon the endometrium. Seventeen cows were fed the orally active progestin melengestrol acetate (MGA) at the rate of 1.0 mg. daily for 14 days for estrus synchroni- zation. The histochemical activity of glycogen phosphorylase and alkaline phosphatase was studied in the endometrium at the time of MGA.withdrawal, on the day of subsequent insemination and 3 days later. Similar histo- chemical determinations were made on endometrial biopsy specimens from naturally cycling cows on the day of estrus at first insemination and 3 days later. The only area.with consistent histochemical activity for both enzymes was the endometrial surface epithelium. Activity was similar in caruncular and intercaruncular surface epithelium. Phosphorylase activity was moderate to heavy throughout the height of the surface epithelium. Alkaline phosphatase activity was confined to a small area in the apical edges of the surface epithelial cells. These patterns were present Lee.Ellis Edwards Jr. regardless of the animal or the day of sampling. The results suggested the influence of estrogen on the endometriumwwas greater than that of pro- gesterone at the times of tissue collection. The similarity of histochemical phosphorylase and alkaline phospha- tase activities in naturally cycling and estrous synchronized cows indi- cates that MBA induced estrus synchronization did not alter the activity of these enzymes in the endometrium at the first estrus after drug with- drawal. The histochemical patterns in~a limited number of animals suggest that conception failures observed in.this thesis were not related-to dif- ferences in phosphorylase or alkaline phosphatase activity at the time of breeding. A HISTOCHEMICAL EVALUATION OF THE BOVINE ENDOMETRIUM By Lee Ellis Edwards Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Large Animal Surgery and Medicine 1971 J 3‘: Jr 6;//a)¢ ACKNOWLEDGEMENTS Many people have-been generous with their time, talents and knowledge and thereby encouraged and enabled me to complete this thesis. The kind counsel offered by members of the Department of Large Animal Surgery and Medicine made my association with them pleasant and worthwhile. The» department chairman, Dr. Fayne Oberst, was always readily available to discuss-problems of any nature, and I appreciate his encouragement. Dr. David Mbrrow served as my academic adviser and donated countless haurs to my graduate program.~ I wish to thank him for his assistance, which included advice on editing this thesis. I.am grateful to Dr. Harold Hafs of the Department of Dairy Physiology for his infectious enthusiasm, which helped motivate my interest in research. He gave me valuable advice on many occasions and aided in the editing of this thesis. Drs. Kenneth Keahey and Stuart Sleight of the Department of Pathology played active roles in this study, and I sincerely appreciate their contributions. A special debt of gratitude is owed to Dr. Robert Kenney of the Uni- versity of Pennsylvania School of Veterinary Mecicine. He contributed greatly to my understanding of histochemical methods and he provided assistance in the interpretation of histochemical sections. Without him this investigation would have lacked considerable motivation and meaning. Drs. Gabel Conner and Bhola Gupta of the Department of Large Animal Surgery and Medicine were most helpful. They provided materials, 11 information and valuable encouragement. Their willingness to help in so many ways is greatly appreciated. Mr. Dennis Armstrong, manager of the Michigan State University dairy herd, was very cooperative in providing experimental subjects. Mr. Robert Wettemann, graduate research assistant in the Department of Dairy Physiology, is to be thanked for allowing me to collect tissues from estrous synchrone ized cows. In conjunction witthr. Harold Hafs, he was also responsible for obtaining the data on ova retrieval reported in this thesis. The patience and helpfulness of Miss Pat Lamb and Mrs. Barbara Wheaton of the Department of Anatomy are gratefully acknowledged. Their efforts and instructions made it possible for me to obtain.frozen sections and conductthe histochemical incubations that made this studypossible. I wish to thank Dr. Rexford Carrow for allowing me to utilize his laborer tory for performing histochemical procedures. I am grateful to Miss Roberta Miler for her efforts in sectioning and staining the formalin-fixed~tissues. Dr. Kenneth McEntee of the New York State Veterinary College was kind enough to help me interpret these sections. I wish to thank Dr. Delbert Whitenack of.the Department of Pathology for his patient instruction, which allowed me to obtain the photomicro- graphs presented in this thesis. I am indebted to the Department of Large Animal Surgery and Medicine for extending the opportunity to pursue graduate studies. Financial support provided by Graduate Research Support Funds from the National Institutes of Health and the Michigan Agricultural Experiment Station made it possible for me to take advantage of this educational opportunity. iii TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE. . . . . . .‘. . . . . . . . . . . . . . . . . . 3 Endometrial Histology. . . . .7. . . . . . . . . . . . . . . . 3 Endometrial Histopatholgoy.. . . . . .-. . . .~. . . . . . . . 6 Endometrial Histochemistry . . . . . . . . . . . . . .'. . . . 10 Histochemical, Biochemical and Endocrinological Relationships. 13 Endometrial Biopsy . . . .-. . . ... . . .'. . . . . . . . . . l7. 'msnmsmnmmons.....-...................19 Experimental Animals . . . ... . . . . . . .>. . . .'. . . . . 19 Sampling Procedure . . . . . . .*. . . . . . . . . . . .v. . . 21 Histochemical Methods. . . . . . . . . . . . . . . . . . . . . 23 RESULTS AND DISCUSSION. . . . . . . . . . . .‘. . . .-. . . . . . . . 26 Histological Observations. . . . . . . . . . . . . . . . . . . 26 Histochemical Observations . . . . . . . . . . . .-. . . .-. . 28- Glycogen phosphorylase. . . . . . . . . . .-. . . . . . 31 Alkaline phosphatase. . . . . . . . . . . . . . . . . . 40 Fertility. . . . . . . . . . . . . . . . . . . . . . . . . . . 48 General Discussion . . . . .-. . . . . . . . . . . . . . . . . 49 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . .-. . . 52 WMCESO s .s s s s o Is 0 s s s s s s s s s s s s s s s s s s 's s s 54 iv LIST OF TABLES Table Page 1 Parity and interval to breeding for naturally cycling and MBA treated cows . . . .'. . . . O O O O O O O O O O O I O O O 20 2 Classification of animals and samples collected for histo- chemical evaluation. . . . . . s s s ‘s s s 0‘. s s s s o s o s 21 Figure LIST OF-FIGURES Page Endometrium. Surface epithelium (1), gland neck (2), peri- vascular leukocytic follicles (3), endometrial glands (4). There is debris in the gland lumens. MGA treated cow (#428), Day 13 of feeding. . . .-. . . . . . . . . . . . . . . . . . . 27 Endometrium. Histochemical phosphorylase activity is not present. Gland lumens are indicated by arrows. Naturally cycling cow (#981), day of insemination. Medium.control section. . . . . .*. . . . . . . . . . . . . . . . . .'. . . . 30 Endometrium. There is no evidence of histochemical alkaline phosphatase activity., A gland lumen is indicated by.the arrow. Naturally cycling cow (#981), day of insemination. Medium control section . . . ... . . . . .‘. . . . . .-. . . .‘ 3O Endometrium. Surface epithelial cells (1) have phosphoryl- ase activity throughout their height. There is minimal histochemical activity in the subepithelial stroma (2). MGA treated cow (#409), Day 13 of feeding. . . . . . .-. . . . 32 Endometrium. Phosphorylase activity is heaviest in the apical portions of some surface-epithelial cells (arrow). MGA treated cow (#1016), day of insemination . . . . .-. . . . 35 Endometrium. The surface epithelium (arrow) has moderate phosphorylase activity. The stroma has light to negative histochemical activity. Naturally cycling cow (#1039), day of insemination... .*. . . .-. . . . . . . . . . . . . . . . . 35 Endometrium. Phosphorylase activity is uniform throughout the height of the surface epithelium, and most intense in the apical portions of the glandular epithelium (arrows). MBA treated cow (#428), third day after insemination . . . . . 37 Endometrium. The phosphorylase activity of the surface epi- thelium.and glandular epithelium is similar to that in Figure 7. Naturally cycling cow (#868), third day after insemination . . . . . . . . . . . . . . . . .'. . . . . . . . 37 Endometrial gland. Phosphorylase activity is represented by the darker areas near the apices of the glandular epi- thelial cells. Naturally cycling cow (#986), third day after insemination . . . . . . . . . . . . . . . . . . . . . . 38 vi Figure 10 ll 12 13 14 15 Page Endometrium, caruncular area. The alkaline phosphatase activity is confined to the distal border of the surface epithelial cells (1). Histochemical activity in the sub- epithelial region is confined to capillary walls (2) and the stroma is not visible. MGA treated cow (#431), Day 13 of. feeding. .~. . . . . . . . . . . . . . . .'. . . . . . . . . . 41 Endometrium, intercaruncular region. The surface epithelium has a band of alkaline phosphatase activity along its apical side.. The stratum compactum has uniform activity (compare ‘with Figure 10). MGA treated cow (#433), day of insemination. 43 Endometrium. The alkaline phosphatase activity of the endo- metrial surface epithelium and stratum compactum is similar to that in Figure 11. Naturally cycling cow (#1041), day of insemination. . . . . . . . .-. . . .'. . . .‘. . . ... . . 43 Endometrial glands. Alkaline phosphatase activity is con— fined to the apical borders of the glandular epithelium. There are variations in activity between adjacent gland cross sections. Naturally cycling cow (#868), day of insemination . . . . . . . . . . .-. . . .7. . . . . . . . . . 45 Endometrium. Alkaline phosphatase activity is restricted to the apical edge of the surface epithelium and glandular epithelium. Stratum compactum and periglandular stroma are positive for histochemical activity.‘ MGA treated cow (#417), third day after insemination . . . . . . .‘. . . .-. .>. .'. . 47 Endometrium. Alkaline phosphatase activity of the surface epithelium is confined to the apical edge. Note similarity to Figures 10, ll, 12 and 14. Naturally cycling cow (#868), third day after insemination . . . . . . . . . .'. . . . . . . 47 vii INTRODUCTION Considerable effort has been devoted to histological characteriza- tion of the bovine endometrium. The normal microscopic anatomy.has been described, and numerous reports deal with pathologic changes in the uterus associated with infertility. The major value of these observa- tions has been their application to the diagnosis of inflammatory changes, resulting from infectious processes in order to determine the effects of these changes upon reproductive function. This histological information is not adequate at present to satisfactorily evaluate the uterus in cases of infertility. Cows with endometritis on.microscopic examination can conceive and subsequently have.normal pregnancies. In addition, a major portion of infertile cows have no microscopic evidence of endometritis. These results emphasize the need for information in addition to morpho- logic changes to evaluate the bovine uterus. Histochemical reactions are indicators of functional status and can provide some of this needed addi- tional information. The hormones involved in reproductive processes act upon their target organs by inducing changes in enzyme activity and distribution. The endo- metrium is a dynamic tissue which undergoes physiologic changes in. response to varying endocrine levels. Although these changes are not easily detectable histologically, they are readily demonstrated by histo— chemical patterns of enzyme localization. The objective of this project was to histochemically monitor 2 endo- metrial enzymes, glycogen phosphorylase and alkaline phosphatase, in 1 2 naturally cycling and estrous synchronized cows. Estrus synchronization was accomplished by feeding an orally active progestin. In estrous synchron- ized cows endometrial tissue was collected prior to the time of progestin withdrawal, at subsequent insemination, and 3 days later. In naturally cycling cows endometrial tissue was collected at insemination and 3 days later for the histochemical demonstration of glycogen phosphorylase and alkaline phosphatase activities. These enzymes are sensitive to fluctua- tions in levels of estrogen and progesterone. Histochemical sections from these animals reflect enzyme distributions in the endometrium as well as endocrine relationships in these animals at the time of sampling. Information related to the lowered fertility in cows at the first insemina- tion following estrus synchronization will be provided by this study. REVIEW OF LITERATURE Endometrial Histology In order to understand pathological changes of the bovine endometrium, the normal histologic.structures must be defined. As stated by Murphey (1924), "there is no fixed anatomical state or condition for any portion of the genitalia; instead, constant changes, cyclic in character, are' occurring." The bovine estrous cycle is normally 21 days in length and has been divided into 4 stages. Proestrus encompasses the 2 or 3 days prior to estrus and is the period of rapid follicular growth or the fol- licular phase.' Estrus lasts 12 to 18 hours.and is the period when the cow‘will accept mating. The next 2 to 3 days are metestrus. Ovulation occurs at the beginning of metestrus, and the corpus luteum begins.develop- ing in this stage. The fourth stage, diestrus, lasts approximately 15 days, encompassing the period of maximum progesterone secretion by the corpus luteum. It is also called the luteal phase. The endometrium. The bovine endometrium consists of a surface epithelium covering a connective tissue stroma which contains glands, blood vessels, and mobile cells (Dawson, 1959). It-may further be divided into caruncular and intercaruncular areas. The caruncles, ovoid projections grossly visible on the uterine lining, participate in the formation of the placenta (Weeth and Herman, 1952; Foley and Reece, 1953). There are normally 75 to 120 present in the uterus (Roberts, 1956). 4 Surface epithelium.' The surface epithelium of the nonpregnant uterus is pseudostratified columnar and is continuous over the caruncles and inter- caruncular areas (Stimson, 1962). The epithelial cell height varies through- out the estrous cycle, according to Weeth and Herman (1952) and Asdell et a1. (1957). It is shortest the second or third day following estrus and. increases gradually to maximum.height during proestrus and estrus. thnson (1965) agreed that it is shortest at 2 to 3 days after-estrus but also stated that the cell length varied greatly within stages of the cycle, making accurate measurements impossible. Dziuk et al. (1958) stated that epithelial height varied more from one location to another in the same section than the range usually accepted for the entire cycle. Marinov and Lovell (1968) observed that the surface epithelium from'heifers had fewer variations in height within the stage of the cycle and more definite changes in height related with stage of the estrous cycle than samples~ from mature cows. The surface epithelium was lowest at estrus and began increasing in height on the fourth day after estrus. Weeth and Herman (1952) reported that the epithelial height was more variable on the caruncles than in the intercaruncular areas. They also noted that the caruncular epithelial cells more frequently degenerated, especially at estrus. Weber at al. (1948) reported a series of changes in the caruncular and intercaruncular luminal epithelium.beginning at pro- estrus and extending into metestrus which were associated with proestrus uterine bleeding. They included formation of vesicles under the epi- thelium, destruction of the epithelium, extravasation of erythrocytes and some tissue damage. These changes were seen principally in.the intercarun- cular areas. Asdell et al. (1957) noted similar events but found slight evidence of tissue destruction and vesicle formation. They described another wave 5 of vacuolization at Day 8 of the estrous cycle. In this study, the histo- logical changes in the caruncular epithelium were similar to those occur- ring in the intercaruncular areas; however, these changes were noted 1 or 2 days earlier on the caruncular surfaces. According to Murphey (1924) and Weeth and Herman (1952), some epithelial cells may be vacuolated at any time, but vacuoles are most numerous during metestrus. Stroma. There is much variation in the density, cellularity and fiber con- tent of the connective tissue stroma. The upper stroma is more cellular and contains many reticular fibers while the deeper region has more collagenous tissue (Trautmann and Fiebiger, 1957). Kingman.(1944) and Skjerven (1956) referred to the relatively narrow densely cellular zone of compact connective tissue just beneath the epithelium as the stratum compactum. The deeper, more loosely arranged collagenous connective tissue was termed stratum spongiosum. These descriptive terms are utilized throughout this thesis. The stratum compactum is relatively thicker in the caruncular areas. The corresponding zone in the intercaruncular endometrium is occupied by many uterine glands (Murphey, 1924; Weeth and Herman, 1952; Foley and Reece, 1953). There is noticeable edema in the stratum compactum at estrus. Asdell et a1. (1957) reported evidence of edema in the deep stroma at 12 days proestrus. It was first noticed in the superficial stroma 4 days before estrus and increased until estrus. The edema rapidly subsided the day after estrus. Skjerven (1956) noted edema in the stratum compactum during 2 stages, a little before and during estrus, and again between days 8 and 12 proestrus. 6 Uterine:glands. The uterine glands are relatively straight and have.their widest lumens in the superficial regions of the endometrium. In the deeper endometrial regions the glands branch and coil. The glandular epithelium is tall columnar and the cell height varies at different stages of the estrous cycle. Johnson (1965) reported that the glandular epithelium is pseudostratified at 17 to 19 days postestrus, while Foley and Reece’(1953) described it as simple.columnar throughout the estrous cycle. Asdell at al. (1957) stated that the nuclei vary in their locations giving a pseudo- stratified appearance which is least apparent from the eighth to the twelfth day postestrus. Glands are numerous in the intercaruncular stratum compactum, and. in this region their necks open into the uterine lumen. No glands are ‘present in the caruncular stratum compactum (Weeth and Herman, 1952). They are present along the sides and at the bases of the caruncles, but are not located in the caruncle proper (Foley and Reece, 1953). The glands exhibit increased branching and coiling during diestrus, and according to Kingman (1944) and Asdell et a1. (1957) the coiling is, greatest between days 8 and 12. Cole (1930) reported that the most distinct histological variation in the cycling endometrium was the change in size and character of the glands. He reported maximum glandular hypertrophy on day 12, followed by regression. Endometrial Histopathology. Most histological abnormalities of the endometrium from infertile cows.are classified under the heading of endometritis. A diagnosis of endometritis is usually based upon microscopic evidence of inflammatory changes which result from infectious processes. .By definition, inflamma- tion limited to the endometrium is endometritis, while involvement of 7 the entire uterine wall is metritis (Jubb and Kennedy, 1970). However, biopsy specimens seldom penetrate deeply enough to include any tissue outside of the endometrium, making evaluation of the entire uterine wall impossible. Thus the definitive division between endometritis and metritis is not always readily established. The literature reveals uncertainty in the evaluation of bovine uterine histologic sections. The lesions of endometritis are not always incompatible with pregnancy, and infertile cows often have no microscopic lesions in their uteri. The lesions of endometritis include diffuse cellular infiltrations of neutrophils, lymphocytes and plasma cells, focal aggregations of lympho- cytes (often termed lymph follicles or nodules), periglandular leukocytic infiltrations, fibrous encapsulation of uterine glands, cystic glandular dilatation, glandular necrosis and replacement of glands by fibrous tissue and inflammatory cells_(Mbss st aZ., 1956; Simon and McNutt, 1957; de 3018, 1961; Dawson, 1961a, 1961b, 1963; Jubb and Kennedy, 1970). The literature justifies the classification of cystic glandular dila- tation with inflammatory changes. However, the terminology is confusing and there is a tendency to.associate cystic endometrial glandular changes with cystic ovarian disease. According to Jubb and Kennedy (1970) the resolution of infectious endometritis may leave a histologic picture cone sisting of cystic uterine glands with periglandular fibrosis. They state that cystic dilatation of the glands is due to periglandular fibrosis. They also describe cystic endometrial hyperplasia as a common lesion in cows with cystic ovarian disease. Affected animals exhibit frequent estrus or nymphomania, and the microscopic lesions are attributed to hyperestrogenism. In advanced cases the degree of glandular hyperplasia may be so extensive that blister-like elevations may be grossly detected on the endometrial surface. It is also emphasized that in some cases of cystic ovaries the 8 endometrium is atrophic rather than hyperplastic with very few dilated glands, These animals usually do net exhibit the external signs of hyperestrogenism that are common in cows with cystic glandular hyper- plasia. Cystic dilatation of uterine glands thus can result from inflam- matory processes and from endocrine disturbances. In a study of reproductive tracts from 77 cows with normal ovaries, Moss et a1. (1956) encountered 5 uteri with cystic glands. They described as typically cystic those glands with wide branched lumens and low cuboidal or flattened epithelium. Some of these glands were surrounded by normal connective tissue while others were enclosed in thick bundles of fibrous tissue. Similar lesions were reported by Frei (cited by Dawson, 1961a), who did not link the occurrence of cystic uterine glands with cystic ovarian disease.‘ This report described infiltration of leukocytes into and around the glands with loss oerpithelium.and periglandular fibrosis. The cystic appearance of the glands was attributed to blockage of the ducts with desquamated epithelium. Simon and MeNutt (1957) reported that about 10% of 109 repeat breeding cows with normal ovaries had cystic uterine glands. In some of them, only one or two cystic glands were. found, and in no case were more than 20% of the glands cystic. Mochow and Olds (1966) found evidence of cystic glandular dilatation in 11.1% of 144 cows. None of the cows had cystic ovaries. The cystic glandular dilatation was attributed to secretory pressure resulting from occluded glands, since the glandular epithelial cells were.flattened and atrophic. They stressed that not all enlarged glands are cystic, since some glands were observed with large lumens and apparently normal epithelial cells. While the above researchers confirmed the presence of cystic uterine glands in the absence of cystic ovaries, Dawson (1961b, 1963) reported a significant connection between these 2 conditions. Cystic glandular 9 change occurred in 29 of 65 cows with cystic ovaries, while only 15 of the remaining 235 cows had similar changes. Inflammation and.cellular infiltrations were present in these cases of cystic endometritis. Other findings emphasize the variations between microscopic changes and clinical performance. Moss et a2. (1956) found histologic uterine lesions in 29 of 77 reproductive tracts from cows with varied breeding records. Although abnormalities were more frequent in cows with histories of reproductive problems, no definite relationships were_established between lesions and fertility.' Included in the study were 2 animals with cystic uterine glands and 7 with encapsulating glandular fibrosis from which viable embryos were recovered as late as 36 days postconception. De Bois (1961) evaluated enddmetrial biopsies from 158 cows and- classified them according to the severity of lesions present! There were advanced to severe lesions of endometritis in 35%. At the time of first insemination, only 5% still had lesions of similar severity. A total of 153 animals was biopsied at first insemination and histological lesions were compared with fertility. 0f the 10 animals in this group that had relatively advanced inflammatory lesions, 4 conceived at first service and 5 conceived at later inseminations. Their average interval from calving to conception was 97 days. In addition, there were 7 animals that did not conceive. Five of these had no microscopic lesions, one had questionable~ changes, and one moderate inflammatory lesions. De_Bois (1961) concluded that inflammatory lesions observed in the endometrium at first insemination resulted from inflammatory processes-occurring early in the postpartum period. He also stated that endometritis is.not often responsible for reproductive failures in clinically normal cows. Similar conclusions were reached by Simon and.MeNutt (1957). They evaluated histological sections from 109 of 200 cows that had been bred 10 at least 4 times and were considered normal except for their failure to conceive. In their opinion, the uteri from repeat breeding cows were histologically similar to those of virgin heifers in most cases. Not more than 15% of the repeat breeding could be attributed to pathological changes in the oviducts or uteri. In-another study, histological sections were prepared from 142 of 150 repeat breeding cows (Graden et aZ., 1968). These animals had been bred at least 3 times without conceiving and showed no detectable abnor- malities on rectal examination. Endometritis was considered a cause of infertility in only 3.32 of these individuals. It appeared that infer- tility rates were lower for cows with cystic glands, endometritis and thin mucosae. A somewhat different view was held by Dawson (1961a), who stated that the economic benefits of the study of histopathology of the uterus are the diagnosis and pregnosis of endometritis. The cows in this study were culled for failure to conceive after each had been bred an average of 6 times. He found severe histologic lesions of endometritis in approxi- mately 50% of 300 reproductive tracts. Endometrial Histochemistry Glycogen. The surface epithelium has predictable variations in glycogen content during the bovine estrous cycle. The cells contain maximum amounts of glycogen beginning 6 days before estrus and continuing until about 7 days after estrus. Conversely, glycogen is either present in trace amounts- or completely absent between Days 8 and 13 of the cycle (Weeth and Herman, 1952; Moss et al., 1954; Skjerven, 1956; Likar and Likar, 1966; Marinov and Lovell, 1968; Larson et aZ., 1970). Skjerven (1956) reported that biopsies taken after fertile inseminations had the same distribution of ll glycogen as those taken on corresponding days of the estrous cycle from cows that were not bred. Although there was some irregularity in the glycogen content of the surface epithelial cells, Moss at al. (1954) reported that the results were uniform in different locations from the same uterus. They also stated that the reactions of the caruncular and intercaruncular epithelium were identical. The stroma has its maximum content of glycogen in the superficial area of the endometrium. The glycogen may be free in the stroma or con- centrated in mobile cells. Moss et a1. (1954) found these cells more numerous in the uterine body.and the caruncles than in the horns. Skjerven (1956) found the most intense reaction for glycogen in parts of the stroma that were most cellular and had dense arrangements of fibers, especially the stratum compactum. It seemed to have a cyclic pattern, reaching peak intensity during the luteal phase. Larson et a1. (1970) found numerous glycogen granules in these cells near estrus. Weeth and Herman (1952) and Likar and Likar (1964) observed little or no glandu- lar glycogen during diestrus. Moss et al. (1954) and Skjerven (1956) maintained that there is no predictable pattern of glycogen distribution in the glandular epithelium. The glycogen content of these cells varied from gland to gland in the same section and from cell to cell within the same glands. Phosphogylase. There are limited observations on the histochemical activity of phosphorylase in the bovine endometrium. Kenney (1964) demonstrated phosphorylase activity in all parts of the uterus, including vessel walls. The surface epithelium was the only area with a detectable cyclic varia- tion. Peak activity was present in the surface epithelial cells during proestrus and estrus. Larson at al. (1970) noted phosphroylase activity mainly in the apical portions of the luminal and glandular epithelial cells. 12 The reaction was most intense at the edge of the surface epithelium adjacent to the uterine lumen. They reported no distinct cyclic patterns in the histochemical distribution of phosphorylase. Alkalinegphosphatase. A predictable cyclic pattern of alkaline phospha- tase localization has been established in the surface epithelium. This reaction is restricted to the apical edges of the epithelial cells. Although there is always some alkaline phosphatase positive material in this location, the reaction is most intense and occupies the_greatest area between Days 9 and 13 of the estrous cycle. Minimal activity is seen in the period a few days before to 5 days after estrus (Moss etyaZ., 1954; Skjerven, 1956;1(enney, 1964; Marinov and Lovell, 1968; Larson et al. , 1970). The stratum compactum.has a heavy.alkaline phosphatase reaction throughout the estrous cycle except in the caruncular areas, where there is no stromal phosphatase activity (Moss et al., 1954; Skjerven, 1956; Kenney,l964; Larson et al., 1970). Moss at al. (1954), Skjerven (1956) and Larson et al. (1970) noted that the alkaline phosphatase activity was always greatest in areas where the stromal fibers were most dense. The periglandular stroma always reacts intensely. Moss et al. (1956) reported that fiber density and consequently alkaline phosphatase activity decreased progressively from the tip of the horns toward the body of the uterus. Weeth and Herman (1952) noted heaviest activity in the stratum compactum at estrus. Moss at al. (1954) and Skjerven (1956) described a tendency for maximum intensity to occur during midcycle and Kenney (1964) stated that there was no cyclic variation in the stromal alkaline phosphatase reaction. The glandular epithelial cells are alkaline phosphatase positive along their apical edges.‘ The upper endometrial glands have the most 13 intense reactions, and activity is decreased in the basal glands (Weeth and Herman, 1952; Moss et aZ., 1954; Skjerven, 1956). While Moss et a1. (1956), Skjerven (1956), and Kenney(l964) report that there is no cyclic variation in glandular alkaline phosphatase activity, Weeth and Herman (1952) recorded an overall decrease in glandular activity at.estrus. The endothelium of capillaries, precapillaries and outer adventitia of many larger vessels are alkaline phosphatase positive (Moss at al., 1954; Skjerven, 1956). These sites do not have cyclic changes in activity. The capillaries are the only alkaline phosphatase positive structures in the caruncular regions. Moss et a2. (1954) and Skjerven (1956) noted a tendency for an inverse relationship to exist between amounts of alkaline phosphatase and glycogen in the locations where these substances are found in the bovine uterus. Histochemical, Biochemical and Endocrinqlggical Relationships Histochemical and biochemical analyses of endometrium from naturally cycling cows and animals treated.with hormones after castration have added to the information relating hormone levels to glycogen content and phos- phorylase and alkaline phosphatase activities. Although a limited number of investigations have been.conducted on cows, some general trends have been established. Glycogen.~ Wrenn et al. (1954) found increased amounts of glycogen in the surface epithelium and upper stroma of biopsies from castrated cows. After these animals had been treated with estradiol benzoate, glycogen disappearEd from the surface epithelium. Very heavy glycogen deposits were seen in the surface epithelium in biopsies from cows that had been given a series of progesterone injections. Biochemical analyses of glycogen (Larson et al., 1970) generally agreed with these histochemical l4 findings. The glycogen content of endometrial biopsies was greatest after castration. Treating the cows with estradiol markedly depressed endometrial glycogen content, while progesterone treatment resulted in intermediate glycogen concentrations. The material utilized contained relatively small amounts of surface epithelium and consisted almost entirely of lamina propria. These results indicated that a steroid balance in favor of estro- gen may be involved in mobilizing glycogen from.the lamina propria. Analysis of a series of endometrial biopsies from cycling cows revealed a tendency for maximum glycogen content during the midluteal phase, but statistically significant differences were not established (Larson et aZ., 1970). Results were determined on a wetdweight basis and may have been influenced by variations in endometrial water content during the estrous cycle. Phosphorilase. Kenney (1964), in a biochemical evaluation of uteri from cattle slaughtered at different stages of the estrous cycle, detected significant cyclic variation in phosphorylase activity with luteal phase diminution. The histochemical pattern in the surface epithelium corresponded to changes in biochemical activity. Results from ovariectomized animals indicated that phosphorylase was responsive to both ovarian steroids, particularly progesterone. He concluded that determinations of endometrial phosphorylase activity have potential as diagnostic aids and indicators of endocrine status. ' Larson et al. (1970) measured phosphorylase activity in biopsies from.cycling and ovariectomized cows. They could not determine distinct patterns of activity or hormone responsiveness for this enzyme.* Alkaline phosphatase.- Both Kenney (1964) and Larson et al. (1970) derived equations for day-of-cycle prediction from variation in biochemical 15 alkaline phosphatase activity during the estrous cycle. The histochemical pattern of the surface epithelium reflected these determinations. Minimum biochemical activity was present in the estrual phase.and following estro- gen treatment of ovariectomized animals. Peak activity occurred during the luteal phase and after progesterone treatment. Kenney (1964) reported the histochemical sections from castrated animals did not correspond to the metabolic determinations from the animals. Wrenn et a2. (1954) studied the histochemical patterns of ovariectomized and steroid treated cows. Castration caused alkaline phosphatase to disappear from the surface epi- thelium and stratum compactum and to decrease in other areas where it normally was present. Following estrogen therapy, alkaline phosphatase. returned to the surface epithelium, increased in the stratum spongiosum and remained absent from the stratum compactum. Progesterone therapy resulted in heavy alkaline phosphatase deposits in the surface-epithelium and its return to the stratum compactum. Because its metabolic activity was reduced at estrus and following estrogen administration, Kenney.(l966) theorized that alkaline phosphatase determinations may help evaluate the influence of estrogen on the endo- metrium. Larson et al. (1970) stated that both estrogen.and progesterone are important-controls over alkaline phosphatase activity in the bovine endometrium. Murdock and White (1968a) measured metabolic activity of endometrial alkaline phosphatase at various stages of the estrous cycle in naturally cycling and estrous synchronized ewes. Alkaline phosphatase-activity followed the growth and regression of the corpus luteum more closely than any of the other 7 enzymes studied, reaching maximum levels during the luteal phase. In another experiment, Murdock and White (1968b) ovariectomr ized ewes and injected them.with ovarian steroid hormones. Progesterone 16 injections significantly increased alkaline phosphatase activity in the endometrium, but estrogen produced no significant effect on alkaline phos- phstase-activity. Estrus synchronization. It has been established that cows have signifi- cantly decreased fertility at the first estrus following estrus synchroni- zation by progesterone treatment (Trimberger and Hansel, 1955; Zimbleman at al., 1970). This reduced fertility is probably the result of hormone imbalances. There is evidnece that uterine metabolic differences exist between the first estrus following progestin withdrawal and naturally occurring estrus. Murdoch and White (1968s) reported higher levels of amylase and alkaline phosphatase activity in endometrium from ewes during the first cycle following progestin induced estrus synchronization than in naturally cycling controls. The ability of the orally active progestin melengestrol acetate. (MGA) to affect the events during the 3 days following insemination is evident in the results obtained by Schul et al. (1969). When MGA was fed to cows beginning 2 days after breeding, their fertility was significantly lower than that of nontreated controls. The fertility of cows fed MGA beginning 4 days after breeding was unaffected. In an evaluation of MBA treated cows, Wordinger et a2. (1970) found lowered endometrial glycogen content on the third day following the first estrus after MGA withdrawal. The plasma progesterone levels were similar in both groups of animals. They attributed the lowered endometrial glyco- gen content in the MGA treated cows to a diminished estrogen influence on the endometrium. 17 Endometrial Biopsy Endometrial biopsy techniques provide useful information in infer- tility research. Because tissues are obtained without slaughter, repeated sampling can be done and an accurate evaluation of fertility is possible by pregnancy diagnosis. The biopsy operation generally has no adverse effect upon the animal. Dzuik et al. (1958) reported that the collection of biopsy samples did not affect the length of the estrous cycle. One cow was subjected to 19 biopsies taken every other day and slaughtered 48 hours after the last sample was taken. There was a slight noninflsmed wound at the site of the last operation and no evidence of scar tissue. from any of the previous operations. In a series of 252 operations on 122 animals by Skjerven (1956), the taking of biopsies in the uterine body or the basal parts of the horns had no significant effect on fertility. Included in Skjerven's study were 13 animals_from which biopsies were taken between Days 7 and 13 after inseminations which later were proved to beefertilel The volume of tissue obtained with the biopsy instrument is small, but previous work indicates the samples are representative, especially for histochemical studies. Moss et a2. (1956) utilized slaughter material from cycling cattle. The alkaline phosphatase and glycogen reactions in. sections from the tips of the horns were similar to those taken near the uterine body in the same individual. Skjerven (1956) stated that a single biopsy is sufficient to obtain an idea of the functional status with histochemical reactions. He reported good agreement between the alkaline phosphatase and glycogen reactions in biopsies taken from different animals at the same stage of the cycle. Kenney (1964) determined biochemical levels of 5 endometrial enzymes. There was no difference in activity between left and right horns and a unilateral effect of the corpus luteum 18 on enzyme activity was not detected. Thus, a biopsy specimen from.one area of the uterus can demonstrate histochemical activities representative of the histochemical patterns in the entire endometrium. MATERIALS AND METHODS Experimental Animals, Estrous synchronized cows. Seventeen primipsrous cows were fed the oral progestin melengestrol acetate (MGA) at the rate of 1.0 mg. per head daily for 14 days. They were injected with 2500 I.U. human chorionic gonada- tropin on the morning of the third day following MGA.withdrawa1, and arti— ficially inseminated on the fourth and fifth day after MGA.withdrsws1. Endometrial biopsies were taken on the thirteenth day of MBA feeding and 2 to 6 hours after the first insemination. Thirteen cows were slaughtered 3 days after the first insemination, and uterine tissues were collected. Endometrial biopsy specimens were collected from the remaining 4 cows on the third day after the first.insemination. Naturally cycling cows. Endometrial biopsies were collected from 15 naturally cycling cows 3 to 6 hours before first insemination on the.day of estrus and again 3 days later. These animals were members of the Michigan State University milking herd. Their reproductive tracts were palpated per rectum.twice weekly and jugular blood samples were collected at 2- to 7-day intervals for future hormone analyses. Formslin fixed and fresh frozen tissues were prepared from these animals. The parity and intervals from parturition to first insemination for these cows is given in Table 1. l9 20 Table 1. Parity and interval to breeding for naturally cycling and MGA COW treated cows i L Number of Interval to Previous First Cow Number of Previous Number Parturitions Breeding (days) Number Parturitions 2:—- Interval to First Breeding (days) 429 716 785' 840 847 850 868 889 967 982 985 986 1021 1039 1048 1 Mean 3 6O 76 107 91 65 64 64, 73 64 58 59 55 68 58 57 68 405 409 417 418 419 420 424 425 428 430 431 432 433 1012 1016' 1020 1035 Mean 1 1 114 92 100 117 89 109 90 104 119 124 112 82 95 107 105 75 __91_. 101 1Naturally cycling. 2MGA treated. 21 SagplingiProcedure~ A total of 77 specimens was taken from 32 naturally cycling and estrous synchronized cows (Table 2). Information obtained by palpation per rectum of reproductive tracts and related physical and behavioral observations were evaluated for each animal to confirm the day of estrus. Table 2. Classification of animals and samples collected for histochemi- cal evaluation Number Alkaline Glycogen. of Cows Phosphatase. Phosphorylsse Sampled Sections Sections MGA treated cows~ Day 13 of feeding 17 13 11 Day of 1st breeding 17 12 10‘ Day 3 after breeding l7 7 8 subtotal ' 51 32 29 Naturally_cycling cows Day of breeding 11. 10 8 Day 3 after breeding 15 13 15 subtotal 26 23 23 Total 77 55 52' It was impossible to obtain satisfactory sections from each biopsy and slaughter specimen due to difficulties with sectioning and unsatisfactory, demonstration of histochemical reactions. These histochemical problems were most prevalent in the sections used for phosphorylase activity. The experience of others indicates that endometrial phosphorylase is histo- chemically capricious (Eranko anvaaklama, 1961). 22 Biopsy technique. Endometrial biopsies were collected with a human rectal instrument.* This device has a universal handle and interchangeable 16- inch stems with hinged-jaw cutting tips. The stems were placed in plastic sheaths and autoclaved prior to each use. The cow's~perineumnwas cleaned, the vulvar lips were parted and the biopsy instrument was introduced into the vagina. The left hand, inserted into the rectum, was used to guide the instrument through the cervix and to determine its location inside the uterus. Biopsies were taken from the uterine body and the basal portions of the horns, as follows: the; instrument's jaw was opened, the uterine wall was pressed into the open end, and the jaws were closed, removing a piece of endometrium. . Handling of tissues. When uterine epithelium is present on a fresh biopsy, it can be seen as a smooth, glistening surface. After-the tissue is frozen, the epithelium cannot be grossly visualized. Therefore, the plane'ofr sectioning must be indicated before freezing the biopsy in order to demon- strate surface epithelium inoa satisfactory percent of sections. Prior to biopsy collection, a 50 ml. beaker containing isopentane was immersed in a dry ice and acetone bath which was held in a stainless steel surgical‘instrument tray. A14 mm. x 75mm. glass test tube-was labelled for each animal to be biopsied and placed in this freezing bath. A precut 5 mm. x 10 mm. piece of filter paper was selected for each sample. It was saturated with water and a small amount of cold gum trsgacsnth was placed in its center. Immediately after collection, the biopsy specimen ‘was carefully removed from the instrument with fine forceps and examined *Yeoman, No. 90-0654, Lawton Surgical Inst. Co., Mbonachie, N.J. V 23 to locate the surface epithelium. It was then placed with the epithelial edge perpendicular to the surface of the filter paper and held in position by gum.trsgacanth.~ Occasionally a specimen.was cut with a razor blade in a plane perpendicular to the uterine epithelium to provide a flat surface that would allow proper positioning on the filter paper. After positioning was complete, the filter paper containing the biopsy specimen was grasped with forceps and dropped into the isopentane. The frozen tissue wasthen placed in a labelled test tube which was sealed with an air—tight rubber stopper. All samples were stored in a freezer at -20 C. The biopsies were stored for a period of one week or less before sectioning. Two endometrial samples were obtained from each of the MGA treated cows at the time of slaughter. The sections were collected from the inter- caruncular regions of the uterine body and basal portions of the horns. One specimen was fixed in buffered formalin and the other was positioned and frozen on a piece of filter paper as previously described. The frozen tissues were stored in tightly sealed 35 mm. film cans in a -20 C. freezer for 3 days and then they were sectioned for histochemical incubations. The formalinized sections were embedded in paraffin, sectioned at 5 u ' and stained with hematoxylin and eosin. Histochemical Methods The frozen tissues were sectioned on a rotary microtome in a Cryostat* at -25 to -30 C. The sections utilized for alkaline phosphatase activity were cut at 811 and those used to demonstrate phosphorylase were cut at 20 u. Sections were lifted from the microtome knife after adhering to *Model CTD, International Equipment Co., Needham Heights, Mass. 24 glass coverslips which were kept at room temperature. Air dried unfixed sections were used in both histochemical procedures. Alkaline phosphatase was demonstrated by using naphthol AS-phosphates as described by Burnstone (1961). The sections were incubated for 15 minutes in a medium consisting of 8 mg. naphthol AS-BI phosphoric acid (sodium salt),* 10 mg. fast red violet LB salt, or 10 mg. fast blue RR salt,* and 10 m1. tris buffer at pH 10. The method described by Eranko and Palkama (1961) was used for phos- phorylase activity.- This procedure is a modification of the method described by Takeuchi-and Kuriski (1955). The incubation was carried out for 60 minutes in a medium containing the following ingredients:. 2 mg. glycogen,* 10 mg. adenosine—S-phosphate,* 100 mg. glucose-l- phosphate (dipotassium salt),* 180 mg. sodium fluoride, 900 mg. poly- vinyl pyrrolidone. (average. M.W. 40,000),** 2 m1. absolute ethanol, 10 ml. 0.1 M acetate buffer pH 5.6, 1 drOp insulin USP (40 I.U./ml.). Native glycogen does not remain in unfixed frozen sections after this incubation (Tskeuchi and Kuriski, 1955). Phosphorylase activity is demonstrated by localizations of_newly formed glycogen within the cells. All incubations were carried out'at 37 C. in freshly made and fil- tered media. Following incubation, the alkaline phosphatase sections were washed for 1 to 2 minutes in distilled water before moudting. The phos-. phorylase sections were handled according to the procedure outlined by Erankfi and Palkama (1961). Glycerine jelly was used as the_mounting medium in all cases. *Sigma Chemical Co., St. Louis, Mo. **Matheson,.Coleman and Bell, Cincinnati, Ohio. 25 Two types of control sections were incubated. The sections for medium control were incubated in media containing all ingredients except the sub- strate. The sections for tissue control were boiled for 30 minutes and incubated in complete media. The phosphorylase sections were stored in closed containers in a refrigerator to minimize fading. The histochemical reactions were inter- preted within 48 hours after the slides were prepared and the results were recorded. Code numbers were used for each section so the identity of the animal fromnwhich the section was taken was not known at the time of interpretation. A second set of interpretations was made at the conclusion of the experiment. At this time, the phosphorylase sections were restained. The slides were held over a beaker of hot water until the coverslips could readily be removed. The tissue sections remained adhered to the coverslips, which were then immersed in.Grsm's iodine solution for 5 minutes. Excess stain and mounting media were removed by washing the sections in distilled water. The coverslips were then mounted on the original slides with glycerine jelly. i1 RESULTS AND DISCUSSION Histological Observations Formslin fixed tissues were not obtained from every animal in this study, which was primarily concerned with histochemistry. The hematoxylin and eosin-stained sections were used for morphologic comparisons. Ana- tomical structures were uniformly defined in these sections compared to the histochemical preparations in which only reacting cytoplasmic components were stained. Eighty-two percent of the histologic sections demonstrated intact surface epithelium. The epithelial surface could not be identified on the formalin fixed biopsy specimens, so the plane of sectioning could not be predetermined. In spite of positioning before sectioning, only 64% of the frozen sections had visible surface epithelium. These results indicate that the handling of specimens utilized for histochemical procedures resulted in loss of surface epithelium. A large number of the gland lumens in these sections contained eosino- philic and basophilic debris. In addition to normal gland secretion, this material contains fragments of glandular epithelial cells and gland secre- tions which were forced into the lumens during the collection of tissue. This regurgitation process is considered a normal event, especially when the tissues are collected with a biopsy instrument. Inflammatory changes were minimal in these sections. There were occasional leukocytic accumu- lations around small blood vessels which were usually located in the stratum compactum (Figure 1). 26 27 Figure 1. Endometrium. Surface epithelium (1), gland neck (2), perivasculsr leukocytic follicles (3), endometrial glands (4). There is debris in the gland lumens. MGA treated cow (#428), Day 13 of feeding (H & E. x 125). 28 Histochemical Observations The histochemical observations recorded during the course of this study were similar to the findings upon reexamination of the sections at the conclusion of the project. The histochemical phosphorylase and alkaline phosphatase activity of the surface epithelial cells was similar in carun- cular and intercaruncular areas. Therefore, these regional-distinctions, are not described in discussions of histochemical results involving the endometrial surface epithelium. All control sections were negative for phosphorylase and alkaline phosphatase activity (Figures 2 and 3). , Gradstions of histochemical phosphorylase activity were estimated by localizations of newly formed glycogen within cells and the intensity of color in these areas. In areas of heavy histochemical activity the reacting cytoplasm was darkly colored and had a homogeneous appearance. In other locations the phosphorylase activity had a patchy appearance and areas of activity were circular spots or sprinklings of color which were interspersed with clear cytoplasm. These areas were sites of moderate or light histochemical activity. The density of the color reaction indicating alkaline phosphatase activity is related to incubation time. The areas of activity become darker as incubation time increases. Although a standard incubation time was used, quantitative judgments of alkaline phosphatase activity were primarily based upon the sizes of areas of positively reacting cytoplasm, rather than the intensity of the reaction in these areas. In both phos- phorylase and alkaline phosphatase procedures, patterns of localization were considered of prime importance in evaluating histochemical activity, and quantitative judgments were made with caution. 29 Figure 2. Endometrium. Histochemical phosphorylase activity is not present. Gland lumens are indicated by arrows. Naturally cycling cow (#981), day of insemination. Medium control section (x 125). Figure 3.‘ Endometrium. There is no evidence of histochemical alkaline phosphatase activity. A gland lumen is indicated by the arrow. Naturally cycling cow (#981), day of insemination. Medium control section (x 250). 31 Glycogen Phosphogylsse In sections from all individuals the stromal phosphorylase activity was highly variable. When activity was present, the reacting fibers were loosely arranged, and reactions had faint to moderate intensity. Activity was usually restricted to the stratum compactum but occasionally included the periglandular stroma. Negatively.and positively reacting areas of. stroma were usually present.in the-same section. Histochemical phosphorylase activity was absent from blood‘vessels with thevexception of reactions in the medial layers of some of the larger vessels. Day 13 of MBA feeding. Sections from 7 cows on the 13th day of MBA feeding had intact endometrial surface epithelium after incubation for demonstration of phosphorylase activity. The histochemical activity of the endometrial_surface epitheliumwwss similar in all individuals on this day. Phosphorylase activity was moderate to heavy.throughout the height of the surface epithelial cells (Figure 4). In the moderately reacting cells, activity was most intense in the apical portions of the cytoplasm. The endometrial glands showed a great deal of variation in histo- chemical activity. Although adjacent epithelial cells within each gland cross section had similar activity, there were differences in enzymatic activity between glands from the same section. There also were differences in activity between cross sections of a single gland. Histochemical phos- phorylase activity was.usually confined to the apical half of the cyto- plasm. There were gradations of density of activity within most glandular epithelial cells, and the areas cf most intense activity were_the apical edges. There was intense activity in the lumens of some of the glands. It is difficult to evaluate the significance of this material. It contains normal gland secretions and fragments of glandular epithelial-cells and gland secretions forced into the lumens by manipulation of tissue. 32 Figure 4. Endometrium. Surface epithelial cells (1) have phosphorylase activity throughout their height. There is minimal histochemical activity in the subepithelial stroma (2). MGA treated cow (#409), Day 13 of feeding (x 250). 33 2§y_of inseminatigg, The surface epithelium.bad moderate to heavy histochemical activity throughout the height of the cells in sections from estrous synchronized cows at first insemination following MGA.withdrawa1 (Figure 5). There were sections with intact surface epithelium from 7 cows. The activity of the surface epithelium was similar in the naturally cycling cows on the day of estrus (Figure 6); however, only 2 of the individuals in this group provided sections with intact surface epithelium. In many.of the moderately reacting cells, activity was most intense in the “1 apical portions of.the cytoplasm. The pattern of histochemical activity in all sections from the day of insemination is similar to that in sur- face epithelial cells from.MGA treated cows on the 13th day of drug g1 administration. The endometrial glands in sections from naturally cycling and estrous synchronized cows on the day of insemination displayed variable histo- chemical activity. The aress of activity were usually confined to the apical 1/2 of the glandular epithelial cells. A few sections from estrous synchronized cows had glands with sites of histochemical activity throughout the length of the epithelial cells. Day 3 sections. Histochemical sections from 8 estrous synchronized and 5 naturally cycling cows had intact surface epithelium. The surface epithelium had moderate to heavy activity throughout the height of the, cells in all animals on the third day following insemination (Figures 7 and 8).‘ This pattern is similar to that observed on the day of insemina- tion and the thirteenth day of MBA feeding. Glandular epithelial cell activity did not differ significantly from previous histochemical patterns (Figure.9). There was much variation in histochemical localization in glands from different animals. In some of 34 Figure 5. Endometrium. Phosphorylase activity is-heaviest in the apical portions of some surfaceepithelial cells (arrow). MGA treated cow (#1016), day of insemination (x 250). Figure 6. Endometrium. The surface epithelium (arrow) has _ moderate phosphorylase activity. The stroma has light to negative histochemical activity. Naturally cycling cow (#1039), day of insemi-. nation (x 125). 35 36 Figure 7. Endometrium. Phosphorylase activity is uniform through- out the height of the surface epithelium, and most intense in_the apical portions of the glandular epithelium (arrows). MGA treated cow (#428), third day after insemination (x 125). Figure.8. Endometrium. The phosphorylase activity of the surface‘ epithelium.and glandular epithelium is similar to that in Figure 7. Naturally cycling cow (#868), third day after insemination (x 125). 37 I I II‘IIIIIUO I-’ll|ll..'t|'lllul--‘OICIIIIIL 38 Figure 9. Endometrial gland. Phosphorylase.sctivity is represented by the darker areas near the apices of the glandular epithelial cells. Naturally cycling cow (#986), third day after insemination (x 500).- 39 the estrous synchronized animals, phosphorylase activity was confined to the apical one-quarter of the glandular epithelial cells. The most notable feature of these results is the similarity in the histochemical phosphorylase activity of endometrial epithelium from all experimental animals. The histochemical activities of surface epithelial cells were the same on the thirteenth day of MGA feeding and the third day after insemination, although 8 days had elapsed.. The histochemical pattern of phosphorylase activity in endometrium from all cows was ' he similar to that described in estrual phase cows by.Kenney (1964); The histodhemical patterns in tissues from Day 13 of MGA-feeding ;F¢v-;_;-.—.i-—A ' and Day 3 following insemination resembled patterns from cows st estrus and insemination in this thesis. These findings probably reflect the effect of estrogen upon the endometrium at the time of sampling. The similarity of histochemical phosphorylase activity in naturally cycling and estrous synchronized cows in this thesis suggests that MGA treatment did not alter the activity of this enzyme in the endometrium at the first estrus following estrus synchronization. However, it is possible that biochemical differences in phosphorylase activity existed that were too small to be detected histochemically. The significance of phosphorylase in the bovine endometrium has not been established. Larson et a2. (1970) did not demonstrate hormonal rela- tionship to phosphorylase activity and were unable to detect any consistent cyclic pattern of activity for this enzyme. Kenney (1964) reported cyclic patterns of histochemical and biochemical activity related to hormone levels. This lack of agreement relative to the activity of phos- phorylase in the normal bovine endometrium andthe histochemical capricious- ness of this enzyme limit its usefulness as an investigative criterion. 4O Alkaline phosphatase Day 13 of MGA feeding, The histochemical alkaline phosphatase activity of the endometrial surface epithelium was similar in biopsy specimens from all animals. Sections from 6 individuals had intact surface epithelium. The histochemical activity was localized in the luminal edge of the surface epithelial cells and extended for approximately l/8 to 1/4 of their length (Figure 10). H The histochemical activity of-the gland necks was the same as that of =‘ the surface epithelium. The remaining glandular epithelium exhibited sig- 3 nificant variation. When activity was present it was seen as a narrow f t ring around the apical margin of the cells. All the cells in a single gland cross section exhibited the same activity. There was a tendency for alkaline phosphatase activity to be the same among glands at the same level of the uterus. The epithelial cells of the superficial areas of. the endometrial glands had larger areas of phosphatase active cytoplasm than those in any other area of the uterus. In the middle and basal regions of the endometrial glands, areas of alkaline phosphatase activity decreased and were represented by thin lines along the apical margins of some glandular epithelial cells, and other glands did not.demonstrste any histochemical activity. Heavily staining alkaline phosphatase positive material was present in the lumens of some glands. This occurred most frequently in glands nearest the uterine lumen. Day of insemination. The alkaline phosphatase activity of the endo- metrial surface epithelium was similar in 4 naturally cycling and 11 estrous synchronized animals from'which sections with intact surface epie theliumwwere.obtained (Figures 11 and 12). It was localized in the luminal 41 Figure 10. Endometrium, caruncular area. The alkaline phosphatase activity is confined to the distal border of the surface epithelial cells (1). Histochemical activity in the subepithelial region is confined to capillary walls (2) and the stroma is not visible. MGA treated cow (#431), Day 13 of feeding (x 500).- 42 Figure 11. Endometrium, intercaruncular region. The-surface epithelium has a band of alkaline phosphatase activity along its apical side. The stratum compactum has uniform activity (compare with Figure 10). MGA treated cow (#433), day of insemination (x.250). Figure 12. Endometrium; The alkaline phosphatase activity of the endometrial surface epithelium and stratum compactum is similar to ‘ that in Figure 11. Naturally cycling cow (#1041), day of insemina- tion (x 250). 43 44 edge of these epithelial cells and extended for approximately 1/8 to 1/4 of their height. The histochemical activity of the glandular epitheliumnwss variable (Figure 13) with the exception of a similarity between activity in the gland necks and surface epithelium. This picture is similar to that~ observed on the 13th Day of MGA feeding. ‘Qayy3 sections. There were histochemical sections with intact sur- face epithelium from 10 estrous synchronized and 9 naturally cycling cows. The histochemical alkaline phosphatase activity of the endometrial sur- face epithelial cells was similar in all individuals on the third day after breeding (Figures 14 and 15). Activity was localized in the apical portions of these epithelial cells in a pattern that did not differ from that observed in endometrial surface epithelium on the day of insemination and the 13th day of-MGA feeding. The glandular epithelium had variable histochemical activity which did not differ from observations on Day 0 and Day 13 of MGA feeding. The stratum compactum and periglandular stroma had moderate to heavy histochemical activity in all sections, regardless of the day of sampling. The vascular activity was confined to the capillaries and precspillsries. These vessels-were most notable in.carunculsr subepithelial regions, where there is no stratum compactum to conceal their identity. They are visible in.cther areas where the endometrial stroma is loosely arranged. No variations in vascular histochemical activity were noted. The histochemical alkaline phosphatase activity of the endometrial surface-epithelium from all individuals in this study conforms to the, pattern previously reported in naturally cycling estrual phase cows (Moss at al., 1954; Skjerven, 1956; Kenney, 1964; Marinov and Lovell, 1968; Larson et al., 1970). The histochemical patterns on Day 13 of MGA 45 L._i_”lro. Figure 13. Endometrial glands. Alkaline phosphatase activity is confined to the apical borders of the glandular epithelium. There are variations in activity between‘ adjacent gland cross sections. Naturally cycling cow (#868), day of insemination (x 125)- 46 Figure 14. Endometrium. Alkaline phosphatase activity is restricted to the apical edge of the surface epithelium and glandular epi- thelium. Stratum compactum and periglandular stroma are positive for histochemical activity. MGA treated cow (#417), third day after insemination (x 125). Figure 15. Endometrium. Alkaline phosphatase activity of the surface epithelium is confined to the apical edge. Note similarity to Figures 10, 11, 12 and 14. Naturally cycling cow (#868), third day after insemination (x 500). 47 48 feeding and Day 3 after breeding were similar to those in endometrial biopsies taken at estrus and insemination in this thesis. Similar histo- chemical activity was observed in castrated cows treated with estradiol. (Kenney, 1964; Larson at al., 1970). The alkaline phosphatase activities observed in this thesis therefore may reflect the influence of estrogen. on the endometrium at the time of tissue collection. The-similarity of the histochemical alkaline phosphatase activities in naturally cycling and estrous synchronized cows indicates that MGA treatment did not alter the activity of this enzyme in the endometrium at the first estrus after drug withdrawal. 'Murdoch.snd White (1968a) reported increased biochemical alkaline phosphatase activity in the endo- metrium of estrous synchronized ewes during the first cycle following estrus synchronization, compared to naturally cycling control animals. If a difference in biochemical alkaline phosphatase activity occurs in the endometrium.from naturally cycling cows at estrus and estrous synv chronized cows at first estrus following estrus synchronization, it was not detected by the histochemical methods used in this thesis. Fertility The fertility of the naturally cycling cows was not satisfactory with only 20% conceiving at first service.' Breeding records show that a low first service conception rate is common in this herd. It is unlikely that the biopsy technique appreciably affected fertility in this project. Other workers have shown that endometrial biopsy specimens are readily obtained without adverse effects on the uterus (Dziuk et al., 1958) or the fertility of the cow (Skjerven, 1956). The uteri obtained at slaughter in this thesis had no significant lesions from prior biopsy operations. 49 The presence of corpora hemorrhagica and corpora lutea in the ovaries from all of the estrous synchronized cows indicated recent ovulations. Upon examination of oviduct flushings from 13 estrous synchronized cows 3 ova were found, all of which had been fertilized. No accurate statement can be made cohcerning the fertility of the animals.fromnwhich ova were. not recovered. There was no explanation;for the low recovery rate of ova. The-4 estrous synchronized cows that were not slaughtered did not conceive at the first insemination after MGA withdrawal. Low fertility was antici- pated at this first service following estrus synchronization. In addition, these animals were inseminated at a fixed time period following MGAuwith- drawal, regardless of the occurrence‘ofestrus. General Discussion The histochemical phosphorylase and alkaline phosphatase activities on the thirteenth day of MBA feeding were similar to the patterns in endo- metrium from estrual phase cows. This suggests a considerable influence of estrogen upon the_endometrium, although the cows had been given repeated daily doses of a progestin and they did not exhibit behavioral estrus until after this drug was withdrawn. The finding of notable estrogen activity in MBA treated cows is supported by the work of Zimbleman and Smith (1966) and Pritchard (1979), who reported that cattle fed.MGA developed higher numbers of large follicles than nontreated animals. Bloss et-al. (1966) theorized that estrogen secreted by these large follicles is responsible for increased gains of body weight observed in MGA treated feedlot heifers,‘ because administration of MBA to steers and spayed heifers did not result. in increased weight gains. The histochemical sections collected on the third day after estrus and insemination in this thesis were similar to those collected at estrus and 50 and may reflect the effects of estrogen on the endometrium. Possibly, histochemical patterns had not adjusted to falling estrogen levels at the time of tissue collection. 0r, progesterone levels may have been too low at this time to exert noticeable effects upon histochemical activity. The similarity of phosphorylase.and alkaline phosphatase activities in estrous synchronized and naturally cycling cows during the 3 days follow- ing insemination indicates that progestin induced estrus synchronization did not alter the activities of these enzymes at the first estrus following drug withdrawal. It is tempting to speculate that reduced fertility at. the first estrus following estrus synchronization is not related to dif- ferences inthe activities of these .2 enzymes. A diminished estrogen influence in estrous synchronized cows, reported by wordinger at al. (1970), was not histochemically detectable.- If the endocrine and enzymatic relationships differed significantly in.the naturally cycling and estrous synchronized cows studied in this thesis, the.histo- chemical methods utilized were not.able to detect.it. The low fertility of the animals utilized in.this thesis did not per- mit adequate histochemical comparison of cows which conceived and failed to conceive. The histochemical patterns in a limited number of fertile animals resembled those from cows which did not conceive, but larger numbers are necessary to validate this observation. There is a great deal of similarity between reported patterns in reproductively normal animals and the histochemical activity of-endometrium from the cows utilized in this study. This suggests that the conception failures observed in this- thesis were not related to differences in-phosphorylase or alkaline phos- phatase activity at the time of breeding. If such differences existed, they were_not detected histochemically. 51 Both histochemical and biochemical methods of enzyme analysis have advantages and limitations. Histochemical reactions-are difficult to quantify, but histochemical patterns of localization and variations in. intensity of histochemical reactions give some idea of quantitative activity.‘ Histochemical methods can also demonstrate specific sites of. metabolic activity.' Biochemical methods are readily quantified but cannot readily provide precise localizations for the activity peaks they detect. The use.of endometrial biopsy specimens for biochemical determinations is complicated by the inability to separate the caruncles from the inter- caruncular tissue at the time of biopsy. The work of Moss at al. (1954), Skjerven (1956), Kenney (1964), and Murdoch and White (1968a, 1968b) has established that the caruncles are metabolically and histochemically dif- ferent from intercaruncular areas. In addition, the relative amounts of epithelial, stromal, and vascular elements vary in biopsy specimens. The use of material collected at slaughter circumvents these problems but this approach is expensive and clinically impractical. The application of histochemical methods to bovine endometrial investigations is in early stages of development. Simultaneous determina— tions of endometrial histochemical and biochemical activities in conjunc- tion with analyses of endocrine levels will provide information on normal interrelationships. This type.of information has significant potential value in evaluation of infertility in clinical and research situations. SUMMARY AND CONCLUSIONS The histochemical activity of glycogen phosphorylase and alkaline phosphatase was monitored in endometrium from estrous synchronized cows. at the time of progestin withdrawal, on the day of subsequent insemina- tion and 3 days later. Estrus synchronization was accomplished by feeding the orally active progestin-melengestrol acetate (MGA). Similar histo- chemical determinations were made on endometrial biopsy specimens.from naturally cycling cows on-the day.of first insemination at estrus and 3 days later. The only area with consistent histochemical activity for both enzymes was the endometrial surface epithelium. Activity was similar in caruncu- lar and intercaruncular surface.epithelium. Phosphorylase activity was moderate to heavy throughout the height of surface epithelial cells in tissues from all experimental animals. Alkaline phosphatase activity of the surface epitheliumnwas minimal. These patterns were.consistent' regardless of the day of sampling. The results suggest the influence of estrogen on the endometriumwwas greater than that of progesterone at the times of sampling. The similarity of histochemical phosphorylase and alkaline phospha- tase activities in naturally cycling and estrous synchronized cows.indi- cates that MGA induced estrus synchronization did not alter the activity of these enzymes in the endometrium at the first estrus after drug withdrawal. 52 53 The histochemical patterns in a limited number of animals suggest that conception failures observed in this thesis were not related to differences in.phosphorylase or alkaline phosphatase activity at the time of breeding. 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