WWW!NWIHNHIWWWWllWNlHHIHHll fr- ‘l‘h" MMfiY Michigan mate University J This is to certify that the thesis entitled ROLE OF OVARIAN FOLLICLES IN LUTEAL REGRESS‘ION OF CATTLE presented by John Leigh Cowley has been accepted towards fulfillment of the requirements for Masters d . Animal Science egree 1n gm Ma or professor Date /fl/é/3; / / MS U is an Aflirmative Action/Equal Opportunity Institution MSU LIBRARIES Egg-25.. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. ROLE OF OVARIAN FOLLICLES IN' LUTEAL REGRESSION 0F CATTLE By John L} Cowley A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1982 ABSTRACT ROLE or OVARIAN FOLLlCLES IN LUTEAL REGRESSION 0F CATTLE By John L. Cowley An experiment was conducted to determine if ovarian follicles, present after midcycle, influence luteal regression in cattle. Four cows had their ovarian follicles destroyed by electro-cautery and X-irradiation on days 10 or ll of the estrous cycle. Six cows, (Sham- irradiated) served as controls. Experiment was terminated on day 22 postestrus at ovariectomy. Absence of visible follicles in X-irradiated ovaries on day 22 postestrus and lower concentrations of estradiol in blood compared to controls led to the conclusion that treatment was efficatious. Basal concentrations of LH in plasma did not differ between groups on any day. By day 22 postestrus concentrations of progesterone in plasma declined in control cows but remained elevated in treated cows. Heights of corpora lutea removed on day 22 postestrus were greater in treated cows compared to controls. Secretion of estradiol-178 was observed to increase prior to decline in secretion of progesterone in blood of control cows. Based on these results, it is concluded that ovarian follicles, present after midcycle are critical to the process of luteal regression in cattle. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION ........................... REVIEW OF LITERATURE ....................... Major Hormones of Reproduction ............... Progesterone ...................... Estradiol ....................... Luteinizing hormone (LH) ................ Follicle stimulating hormone (FSH) ........... Corpora Lutea of the Estrous Cycle ............. Role of the CL in cyclicity .............. Formation of the CL .................. Regulation of Luteal Function ................ Regression of Corpora Lutea- ................ Role of the Uterus in Luteal Regression ........... Prostaglandins and Luteal Regression ............ Oxytocin and Luteal Regression ............... Estrogens and Luteal Regression ............... Progesterone and Luteolysis ................. Mechanism of Action of PGFZa ................ Blood Flow ......................... Role of Lysosomes in Luteal Regression ........... Effects of PGF a on Metabolism in Luteal Cells ....... Role of Develoament of Follicles in Luteal Regression. . . . Luteal Function in the Absence of Ovarian Follicles ..... Effects of X-irradiation on Ovarian Tissue ......... Summary of Review of Literature ............... MATERIALS AND METHODS ....................... General ........................... Animals ........................... Surgery ...... _ ..................... Treatment ......................... Sampling of Blood ...................... Quantification of Hormones ................. PGF (1 Ova; Statistical Analysis .................... iécéonyIIIIfIIIIIIIIIIIIII ...... —l DONme-wa w RESULTS ............................. Efficacy of Treatment ................... Effects of Treatment on Concentrations of LH in Plasma. . . Effect of Treatment on Luteal Function ........... Relationship of Secretion of Estradiol-l73 to Progesterone. Relationship of Estradiol-178, PGF a and Progesterone Characterized in Individual Cow; . ........... DISCUSSION ............................ LIST OF REFERENCES ........................ "ii Table 1. Table 2. LIST OF TABLES Concentrations of Estradiol-l78 in plasma and inventory of follicles prior to and following X-irradiation or sham-irradiation ......... 35 Plasma progesterone and weights of corpora lutea of cows on day 22 postestrus, following X-irradiation on day 10 ............. 4l LIST OF FIGURES Figure l. , Effect of sham-irradiation or X-irradiation of ovaries on concentrations of estradiol-l78 in plasma from utero-ovarian veins draining both ovaries. Values represent daily mean concen- trations + s.e. of all animals within treatment group. J‘signifies time of treatment ....... 36 Figure 2. Effect of follicle cautery and X-irradiation or shampirradiation on concentration of LH in jugular venous plasma. Values represent daily mean concentrations + s. e. from all animals within treatment group. / signifies time of treatment ................... 39 Figure 3. Concentrations of progesterone in jugular venous plasma of cows following electrocautery of follicles, X-irreadiation of ovaries or sham-irradiation ................. 43 Figure 4. -Concentrations of estradiol-l73 in plasma from the utero-ovarian vein draining the ovary bearing a corpus luteum and progesterone from the jugular vein in five sham-irradiated cows which underwent luteal regression ......... 45 Figure 5. Concentrations of estradiol- l7B and PGF a in plasma from the utero-ovarian vein draihing the ovary bearing a corpus luteum and proges- terone from the jugular vein in cow number 6493. ....................... 4B Figure 6. Concentrations of estradiol- l7B and PCP a in plasma from the utero-ovarian vein draihing the ovary bearing a corpus luteum and progesterone from the jugular vein in cow number 6592 ..... 50 Figure 7. Concentrations of estradiol-178 and PCP a in plasma from the utero-ovarian vein draifiing the ovary bearing a corpus luteum and progesterone from the jugular vein in cow number 6585 ..... 52 Figure 8. Hormones that influence luteal regression in cattle: A model of the bovine estrous cycle. . . . 64 INTRODUCTION Efficient production is a prime goal in agriculture. In animal agriculture, reproduction is critical to production. Animal scientists have provided many tools necessary to improve reproduction, yet much remains to be learned. Genetic improvement, a hallmark of the dairy industry for 30 years, is based on selection for desireable traits the most important being high milk production. .Although reproduction is a prerequisite for milk pro- duction, reproductive performance is not one of the traits generally selected for. Yet, failure to reproduce is a primary reason for culling. Often the high milk producing cows are retained in herds in spite of failure to conceive within a desired period of time. Incentives to main- tain these cows in the milking herd are great. Better understanding of the physiological process of reproduction is the means by which animal scientists will devise the tools necessary to deal with infertility in todays dairy industry. Critical to fertility in cows are expression of estrus and time ovulation related to estrus. These occur following cessation of produc- tion of progesterone by the corpus luteum (luteolysis). Defects in the luteolytic process could influence fertility. Important to understanding the process of luteal regression (luteolysis) is determining factors which initiate this event. It is the intent of this study to provide further insight into factors influencing luteal regression. REVIEW OF LITERATURE Factors associated with regression of corpora lutea in cattle and sheep will be discussed in this review. Although cows are the intended focus, the abundance of literature concerning reproduction in ewes and many apparent similarities between ewes and cows justify using data from ewes to support the bovine model. Data concerning other species will not be discussed except to clarify or support important concepts not tested in cows or ewes. With each estrous cycle in cows there is opportunity during estrus for conception. In the absence of pregnancy cyclicity continues providing more opportunities for conception. Most cows undergo estrous cycles every 17 to 25 days with an average cycle of 21 days (Moeller and VanDemark, 195l; Asdell gt $1,, l949). A corpus luteum (CL) forms with each estrous cycle and as a transient endocrine organ is a major component affecting estrous cycles. Continued luteal function is necessary for maintenance of pregnancy; however, in the absence of pregnancy, renewal of cyclicity is dependant upon regression of corpora lutea. The primary function of corpora lutea is secretion of progesterone a major reproductive hormone. Proges- terone, in concert with other hormones of reproduction, influences the progess of reproduction. Major Hormones of ReprodUction Progesterone: As luteal tissue forms during metestrus and diestrus 3 concentrations of progesterone circulating in the blood increase. Herriman gt_al, (1979) reported plasma concentrations of progesterone to rise in a linear fashion from day 2 postestrus through day 10. Netteman gt 31. (1972) reported circulating concentrations of progesterone to increase between days 3 or 4 and day 11 postestrus. From day 10 post- estrus through day 16 concentrations of progesterone in blood fluctuate but remain elevated. By day 18, concentrations of progesterone decline as the corpus luteum regresses (Hetteman gt 31,, 1972; Chenault gt_al,, 1975; Garverick gt al., 1971). Absolute concentrations of progesterone observed by various groups often differ most likely due to variation between assays, however, the patterns of secretion of progesterone were similar. On day 2 postestrus progesterone in blood increases through days 8 to 10, is maintained through days 16 to 18 when luteolysis ensues, progesterone in blood then declines to basal levels observed throughout estrus. Estradiol: In cows, estradiol in the blood is difficult to quantify during most stages of an estrous cycle. For this reason changes in con- centrations of estradiol during diestrus are variable and difficult to assimilate. Hetteman gt 31. (1972) quantified estradiol in single serum samples taken from heifers on days 2, 4, 7 and 11 postestrus and reported values between 3.0 p9/ml and 3.9 DQ/ml with no appreciable variation over time. Significant increases in concentrations of estradiol occurred during proestrus and estrus. Shemesh §t_gl, (1972) measured estradiol in blood of nine cows throughout their estrous cycles and observed three periods of increased concentrations of estradiol. The greatest increase was associated with estrus and smaller increases were noted on days 4 and 11 postestrus. Dobson and Dean (1974) reported a small rise in estradiol around day 4 postestrus but did not observe an increase at midcycle. Hansel and Ecternkamp (1972) and Glencross £3.31, (973) observed high concentrations of estradiol at estrus and another increase during metestrus, but very low concentrations at midcycle in postpartum cows. However, concentrations were noted to increase on day 12 postestrus and remained slightly elevated until proestrus when concentrations of estradiol increased markedly concomitant with the decline in progesterone. In summary, researchers agree that the greatest concentrations of estradiol occur at proestrus and estrus with a less dramatic increase during metestrus. There is equivocal evidence that concentrations of estradiol increase during mid to late diestrus. However, frequency of sampling, preparation of blood samples, site of sampling and limitations of assays in reports cited above, may account for variable tests of this concept. Luteinizing hormone (LH): Relatively low (baseline) concentrations of LH are observed throughout most of a bovine estrous cycle. Mean con- - centrations of LH in blood range between 1 and 2 ng/ml during diestrus (Netteman_gt;gj, 1972). Rahe gt;§l, (1980), aware that LH is character- isticly released in a pulsatile fashion, quantified LH in blood of four heifers on days 3, 10, and 19 postestrus. Observations were that concen- trations of LH in blood fluctuate in a pulsatile fashion that varies with stage of an estrous cycle. During metestrus (day 3 postestrus), pulses of LH were of low amplitude (ALH = .3 to 1.3 ng/ml) and high frequency (20 to 30 pulses/day). The mean concentrations of LH varied among animals (1.3 ng/ml to 2.2 ng/ml). At mid-diestrus (day 10 postestrus) 'pulses of LH were characterized at high amplitude (ALH = 1.2 to 7.0 ng/ml) and low frequency (6 to 8 pulses/day). Again, mean concentrations of LH differed among animals on day 10. Mean concentration of LH on day 10 postestrus did not differ from mean on day 3. Thus, there is no change in basal concentrations of LH between day 3 and day 10, however the pat- tern of secretion of LH did differ. Sources of variation and potential biological effects of differences in secretory patterns of LH between days 3 and 10 postestrus are not known.. Frequency of pulses of LH increase as the preovulatory surge of LH approaches. The preovulatory surge of LH lasts from 5 to 10 hours and is composed of numerous episodic peaks high in magnitude (Rahe gt_al,. 1980; Chenault 33.31,, 1975). Follicle stimulating hormone (FSH): A definitive study aimed to adequately characterize the pattern of circulating concentrations of FSH in cows has not been dbne. Increased concentrations of FSH occur in blood at the time of the LH surge during proestrus (Roche and Ireland, 1981; Dobson, 1978). Decreased concentrations of FSH were found in pit- uitary glands during this same interval (days 18 through 20) (Hackett and Hafs, 1969). However, a depletion of FSH in pituitaries was also noted to occur at midcycle (Desjardens and Hafs, 1968). Although this observation supports the possibility that secretion of FSH into blood increases at this time, no direct evidence exists. Although the LH and FSH surges associated with proestrus occur concomitantly, the secretory patterns of FSH do not always parallel patterns observed for LH (Dobson. 1978). Concentrations of FSH in blood do vary during metestrus and diestrus, however discrete patterns are not yet readily discernable (Akbar ethal,, 1974). With the advent of better assays and knowledge that acute changes in secretion of pituitary hormones contribute to the overall hormonal patterns, the nature of secretion of FSH in cattle will become known. Corpora Lutea of the Estrous Cycle Role of the CL in cyclicity: The primary role of the corpus luteum is to produce progesterone. Significant variations in concentrations of progesterone in blood affects estrous cyclicity; Early regression of corpora lutea results in shortened cycle length. Prolongation of luteal function results in longer than normal estrous cycles. In cows and ewes, concentrations of progesterone in blood rise during metestrus as the CL develops. Concentrations of progesterone remain elevated throughout diestrus and decrease in late diestrus-early proestrus (Wetteman gt 31,, 1972). Estrus and ovulation occur spontan- eously only after luteal regression and production of progesterone ceases. Continuous administration of progesterone prevents estrus and ovulation in cows (Christian and Casida, 1948) and ewes (Dutt and Casida, 1948) thus prolonging estrous cycles. Withdrawal of exogenous progesterone is followed by estrus and ovulation in cattle (Roche, 1976) and sheep (Dutt and Casida, 1948). Enucleation of corpora lutea at midcycle in cattle results in premature estrus and ovulation (Anderson gt 31,, 1965; Snook gt 31,, 1969; Hobson and Hansel, 1972). Therefore, relative consistency of normal estrous cycle length is in part dependent on interval of luteal function. There have been numerous studies attempting to elucidate mechanisms by which corpora lutea and/or progesterone influence estrous cycles. In cycling cows and ewes regression of corpora lutea is followed by rapid development of follicles and preovulatory surge of LH (Chenault gt.al,. 1975). This sequence was also noticed following enucleation of CL in cattle (Hobson and Hansel, 1972) and in sheep (Karsch gt 91., 1979); Dvariectomy during the luteal phase of an estrous cycle results in in- creased concentrations of LH in sera of cattle (Hobson and Hansel, 1972) and sheep (Butler gt_gl,, 1971). This is termed the Apost castrational rise in LH" and does net achieve the magnitude of the preovulatory LH surge. Replacement of endogenous progesterone following ovariectomy with progesterone implants reduces magnitude of the post castrational rise in LH (Beck gt al,, 1976). The ability of progesterone to prevent a surge of LH and ovulation following removal of corpora lutea in sheep was dem- onstrated by Karsch gt 11. (1979). Exogenous progesterone blocked estradiol induced surges of LH in ovariectomized ewes (Howland £3 91,, 1978). Kesner (1981) showed that progesterone is an effective blocker of LH and FSH surges in cattle. Therefore, the LH surge does not occur during a period of luteal function. Karsch gt 31. (1979) suggested that the slight increase in concen- tration of LH following luteal regression might stimulate increased secretion of estradiol. In the absence of progesterone, exogenous estradiol is capable of eliciting a LH surge in ewes (Hobson and Hansel, 1972) and in cattle (Beck and Convey, 1977; Short gt_gl,, 1979). However, Fogwell gt gl_(l978) observed increased concentrations of estradiol to occur in blood of heifers following removal of CL whether or not increased secre- tion of LH was blocked by anesthesia. Thus, the small increase in con- centration of LH observed just following luteal regression may not be important to increased secretion of estradiol thought to stimulate the LH surge. Clearly, corpora lutea do influence events associated with cyclicity primarily through production of progesterone. In order for estrus and ovulation to occur and cyclicity to resume, the corpus luteum must regress. Although timing of luteal regression influences lengths of estrous cycles, additional evidence indicates timing of formation of corpora lutea is also important. Administration of progesterone from estrus through metestrus shortened estrous cycle lengths in cattle and sheep (Woody 391., 1967). Formation of the CL: Following ovulation, granulosa and theca cells of an ovulated fbllicle differentiate into luteal cells. Luteal cells derived from the theca interna dominate the population of luteal cells observed by day 7 postestrus.. Luteal cells derived from granulosal cells are prominent and dividing up to day 4 postestrus. Luteal cells derived from the theca interna appeared to retain ability to respond to LH by proliferating after day 4 when granulosa luteal cells have ceased mitotic activity; Mitosis of luteal cells is complete by day 8 postestrus but hypertrophy of existing luteal cells and proliferation of connective tissue may continue for 3 more days (Rajakoski, 1960). This period (days 8 through 11) was characterized by increased ability to bind LH, ~increased secretion of progesterone, increased adenylate cyclase activity and increased luteal weight (Fitz gt al., 1980; Erb gt_gl,, 1971). Regulation of luteal function: Hypophysectomy of ewes during the luteal phase of an estrous cycle results in loss of luteal function (Kaltenbach gt 31,, 1968; Hixon and Clegg, 1969). Continuous infusion of pituitary extract prevents loss of luteal function following hypophy- sectomy (Kaltenbach £3 31., 1968). However, purified pituitary extract high in LH activity required concurrent administration of prolactin in order to maintain luteal function in hypOphysectomized ewes (Denamur gt ‘31., 1973). Administration of antisera to bovine LH during the period of luteal development decreased luteal weights and concentration and 10 content of progesterone of corpora lutea. However, the same treatment during late diestrus resulted in lengthening of the estrous cycle. Hhile LH is critical for luteal development it may also be involved in luteal regression due to support of follicular development (Snook gt_al,, 1969). In 31352, LH has been demonstrated to be the primary luteotropin of ewes (Kaltenbach gt a_1_. , 1967) . ConCentrations of progesterone in blood have been shown to fluctuate during diestrus yet no alteration in concentrations of LH have been assoc- iated with these changes (Rahe £3 31,, 1980; Roche and Ireland, 1981). However, techniques in handling, storing and processing blood samples can alter assayable concentrations of progesterone (Vahdat gt 31,, 1981) and account for some variation. Changes in numbers of receptors or binding characteristics for LH on luteal cells may account for variation in secretion of progesterone. ~However, Diekman gt al, (1978a) reported no change in affinity of receptors for LH on luteal cells during diestrus in ewes. No significant changes in numbers of receptors occurred during mid-diestrus. However, comparisons were of data pooled within day and may have masked transient changes. Suter 3; 11. (1980) demonstrated that large doses of LH increased total numbers of receptors for LH in luteal tissue associated with increased production of progesterone in ewes. ‘ Receptors for LH associated with lysosomes were demonstrated to have similarities to those on plasma membranes. However, because some characteristics were different, it was suggested that these receptors were of different origins (Rao gt_a13, 1981). The possibility that differences in characterisitics.were due to the dynamic process of receptor degradation by lysosomes was not tested. Characterization of 11 receptors for the luteolytic substance prostaglandin an (PGFZa) in luteal tissue of cows have been reported. Compared to other subcellular fractions, there were greater numbers of receptors for PGEI, PGan and LH in both lysosomes and plasma membranes of luteal cells (Mitra and Rao, 1978). Binding of PGan to lysosomal and plasma membranes increased from days 3 to 20 postestrus then declined by days 21 to 24 in cows. However, competitive binding characteristics of receptors for hCG were not different on days 13 and 20. Concentrations of progesterone in plasma were higher on day 13 postestrus than day 20. Thus, although a temporal association existed between secretion of progesterone and binding of PGan to luteal cells, acute or chronic cause effect relationships have not been tested (Rao 3511., 1979). Exogenous estradiol causes luteal regression in cows (Kaltenbach _t_ 31., 1964; Greenstein _e_i_:a_1_., 1958) and ewes (Warren g; _a_1_., 1973; Stormshak gt 21,, 1969). Variations in circulating concentrations of estradiol have been recorded in cows during diestrus (Shemesh gt al,. 1972; Hansel and Ecternkamp, 1972; Dobson and Dean, 1974) and ewes (Scaramuzzi 3311., 1970; Hauger Jig” 1977). However, a direct relationship between changes in concentrations of estradiol and pro- gesterone has not been demonstrated. Sheridan gt a1, (1975) did demonstrate variations in uptake of estradiol into luteal tissue during the luteal phase of the ovine estrous cycle. To date, there is no evidence that acute or transient changes in receptor characteristics of traphic or 1ytic substances occur during diestrus in cycling cows or ewes. It is possible that changes in concentrations of gonadotropins and luteolysins in blood interact with transient changes in their receptors to influence luteal function. This theory would be hard to test because it is likely 12 that each animal differs slightly in those parameters such that pooling data from different animals would result in no significant change in pattern. The mechanism by which progesterone is secreted from corpora lutea may influence fluctuations in circulating concentrations of progesterone in blood. Progesterone was shown to be characteristically packaged into distinct secretory granules within luteal cells and released by exocytosis (Genmell £11., 1974; Hillcox and Alison, 1982; Quirk _e_1_:__al., 1979). Sawyer gt 31, (1979) reported a relationship between formation and release of secretory granules and the secretory pattern of progesterone during the ovine estrous cycle. Furthermore, formation and release of secretory granules lghyitrg was enhanced by the addition of LH. Nillcox and Alison (1982) reported the existence of a specific binding protein for proges- terone within luteal cells. Condon and Pate (1981) found addition of serum to potentiate release of progesterone, independent of synthesis, from bovine luteal cells ighyitrg. Thus, a factor contained in serum may influence secretion of progesterone. Adenosine has been shown to poten- tiate LH induced secretion of progesterone by rat luteal cells in yitrg. Adenosine also antagonized the depressant effect of PGFZa on production of progesterone (Behrman _t._l., 1982). In summary, luteal function is dependant upon gonadotropic support, however, variations in circulating concentrations of progesterone do not appear to be due to changes in secretion of pituitary gonadotropins. Changes in packaging and release of progesterone as well as influences of luteolytic factors or balance of luteolysins and luteotropins may account, at least in part, for variations in secretion of progesterone. Additionally, collection, handling and storage of blobd samples may 13 account for some variation of results of hormonal assays. Regression of Corpora Lutea Luteal regression involves a complete loss of production of proges- terone (functional regression) and degeneration of luteal tissue (structural regression). Regression of corpora lutea begins around day 16 postestrus in cattle and around day 14 postestrus in ewes (Hansel 53.31,, 1973). Donaldson and Hansel (1965) reported early aspects of luteal regression were associated with loss of cytoplasmic stippling and rounding of cell outline. Vacuolation was observed at the cell periphery followed by cytoplasmic condensation and pyknosis. Gemmell gt 31, (1974, 1976) reported decreased numbers of densely staining granules associated with decreased secretion of progesterone and appearance of autophagocytic bodies within luteal cells as the first morphological signs of luteolysis. These changes were observed as early as day 12 of an ovine estrous cycle. By day 15 postestrus degradation progressed to a point were cellular organization was lost. Lysosomal activity appeared increased and accumu- lation of lipid droplets were observed. The dramatic increased degenera- tive changes was temporally associated with final decline in concentrations of progesterone in blood of cycling ewes (Thorburn and Mattner, 1971). McClellan gt_al, (1977) reported lysosomal activity to increase in luteal tissue by day 15 postestrus in ewes. Dingle gt a1. (1968) reported total lysosomal enzyme content in corpora lutea of ewes not to change throughout diestrus, but fragility and enzyme activity of lysosomes increased during late diestrus. Rao gt 11. (1979) reported reduced binding of gonadotropin to bovine luteal membranes to occur after concentrations of progesterone 14 had declined in blood. However, Diekman t al. (1978a) and Spicer gt_al, (1981) reported a loss of luteal receptors for LH occurred concomitantly with luteal regression in ewes and cattle. In bovine corpora lutea, binding of PGFZa increased during late diestrus (Rao £3 21,, 1979). Role of the Uterus in Luteal Regression Hysterectomy during midcycle prolongs lifespan of corpora lutea in cattle (Hiltbank and Casida, 1956; Brunner gt al., 1969; Anderson gt 11., 1962), in ewes (Hiltbank and Casida, 1956) and in many other mammalian species (Melampy and Anderson, 1968). In most studies the luteolytic effect of the uterus has been shown to be generated from the uterine horn adjacent to the ovary bearing a CL in both cows and ewes (Moody and Ginther, 1968; Ginther gt a1,, 1967; Ginther, 1967; Mapletoft gt 91., 1975). Integrity of vasculature between ovaries and uteri was necessary for luteal regression (Dobrowolski gt 31,, 1970). Vascular anatomy of sheep and cattle, species in which the uterine luteolytic effect is unilateral, had ovarian arteries in close apposition to ovarian veins. This arrangement was not evident in mares, an animal in which the luteo- lytic effect of the uterus is not local (DelCampo and Ginther, 1973; Ginther and DelCampo, 1974). Blood taken from uterine veins of ewes on day 15 postestrus caused premature luteolysis when infused into arterial supply of autotransplanted ovaries (McCracken gt_al,, 1972). Although the uterus is important for luteal regression, it may not be the only component or variable. In primates, luteal regression is independent of the uterus and is controlled principally within the ovary 15 (Karsch and Sutton, 1976; Auletta gt 91,, 1978). In addiiton, hysterectomy does not consistently prolong CL in cows (Ward gt_al,, 1976; Brunner gt 21,, 1969; Anderson gt_al,, 1962; Hansel and Seifart, 1967). Therefore, control of luteal regression in cows may be influenced by ovarian as well as uterine factors. Prostaglandins and Luteal Regression Prostaglandin an induced luteal regression in pseudopregnant rats (Phariss and Hyngarden, 1969) and in most other mammals tested (see Horton and Poyser, 1976). Concentrations of PGFZa have been observed to increase in utero-ovarian venous plasma of ewes during the period of luteal regression (Bland 2!;él:9 1971; Thorburn gt_al,, 1972). Concen- trations of prostaglandin forming cyclooxygenase in ovine endometrial tissue increased during the period of luteal regression (Huslig gt 11., 1979). Increased content of PGFZa in uterine endometrium and increased concentration of PGan in ovarian venous blood occurred during the period of luteal regression in heifers (Shemesh and Hansel, 1975a). McCracken gt_gl, (1972) reported infusion of PGan into an ovarian vein of a ewe resulted in preferential transfer to the adjacent ovarian artery. Interestingly, first detection of veno-arterial transfer of PGan was delayed relative to onset of infusion but transfer was sustained for 90 minutes following cessation of infusion. Hixon and Hansel (1974) reported peak concentrations of PGan in ovarian arterial blood of heifers to occur 40 minutes after infusion into a uterine lumen. Episodes of increased concentrations of PGan in blood from both uterine and utero-ovarian veins of ewes were not always temporally correlated with each other or with progesterone and thus implicating a 16 potential ovarian source of PBan (Nett g§.al,, 1976). Hansel _t“al., (1976) demonstrated that bovine luteal and follicular tissues can synthe- size PGF in vitro. Injection of PGFZa into the largest follicle on the ovary bearing a CL caUSed premature luteal regression in ewes (Inskeep gt;al,, 1975) and heifers (Fogwell gt_al,, 1978). The mechanism of transfer of PGan from follicle to CL involved a local (unilateral) yet extraovarian pathway (Fogwell gt;al,, 1977).. These observations raise the possibility that in addition to the uterus, ovarian factors may contribute to luteal regression. Oxytocin and Luteal Regression Administration of oxytocin early in an estrous cycle results in premature.lutea1 regression (Hansel and Wagner, 1960; Anderson gt_al,. 1965; Black and Duby, 1965). This luteolytic effect of oxytocin is dependant upon presence of the uterus (Ginther gt_al,, 1967; Anderson gt al,, 1965). Administration of atropine or epinephrine inhibited the luteolytic effect of oxytocin but did not affect spontaneous luteal regression (Black and Duby, 1965). However, ewes immunized against oxytocin exhibited prolonged estrous cycles (Sheldrick gt_al,, 1980). Newcomb gt_al, (1977) showed that injection of oxytocin early in the bovine estrous cycle resulted in increased concentrations of PGF in the posterior vena cava. This effect was not observed when oxytocin was administered after day 4 postestrus. In ewes, oxytocin injected on days 4 and 14 postestrus caused increased uterine secretion of PGan but not on day 8postestrus (Roberts and McCracken, 1976). Circulating concentrations of estrogen have been reported to be increased on days 3 and 14 but not day 8 17 of the ovine estrous cycle (Hauger gt 31,, 1977). In anestrous ewes, oxytocin augmented estradiol induced increased release of PGF but in the absence of estradiol, oxytocin was without effect (Sharma and Fitzpatrick, 1974). Although inconclusive, these results are consistent with the concept proposed by Roberts and McCracken (1976) that estrogen and oxytocin interact to cause increased release of PGFZa for the luteolytic process. Estrogens and Luteal Regression Administration of estrogen to cattle after midcycle causes premature luteal regression (Niltbank gt 31,, 1961; Niswender 33.21:’ 1965; Brunner ‘gt al,, 1969). However, estrogen administered early in the estrous cycle has variable effects on luteal regression (Greenstein gt_al,, 1958; Loy gt 31,, 1960; Kaltenbach gt 21:: 1964). Administration of estradiol on days 1 through 3 did not alter lengths of estrous cycles (Ginther, 1970). Piper and Facts (1965) reported estrogen given daily starting on day 4 postestrus extended luteal life span in ewes. Administration of estradiol on days 4 through 7 of the ovine estrous cycle lengthened cycles. Howland gt a1, (1971) reported surges of LH to occur in blood of ewes given a single injection of estradiol on day 4 but not day 11 postestrus. Thus, lengthening of estrous cycles following administration of estradiol early in estrous cycles may be due to increased luteotropic support. Moreover, estradiol may not provide any luteolytic influence without prior pro- gestational "exposure". Administration of progesterone during metestrus facilitated estradiol-induced luteolysis on days 5 and 6 of the ovine estrous cycle (Warren gt_al,, 1973).. Hysterectomy prevents the luteolytic effect of estrogen in ewes 18 Bolt and Hawk, 1972; Stormshak _tn_l., 1969; Chakraborty and Stormshak, 1976). Although, Kaltenbach gt 31, (1964) and Brunner gt_al, (1969) reported reduced luteal weights following treatment of hysterectomized heifers with estrogen, the uterus was necessary for luteal regression (Brunner gt_al,, 1969). Estradiol did however, facilitate luteolysis induced with PGan in ewes (Gengenbach £3 31,, 1977). Thus, both uterine and extra-uterine factors may influence luteal regression induced with estrogen in cattle and sheep. Duration of treatment with estrogen may be more important to luteolysis than dose of estrogen administered. Luteal weights were reduced further by infusion of low amounts (10 ug/hr) of estradiol for 24 hours than by higher doses (41 ug/hr) for 12 hours. However, treat- ment for 48 hours was no more efficacious than 24 hour treatment (Bolt, 1974). Infusion of estradiol into uterine arteries of ewes for 6 hours on day 14 postestrus resulted in increased secretion of PGan into uterine veins but not days 6 or 10 postestrus. However, Ford gt_al, (1975) detected increased secretion of PGF into uterine venous blood on day 11 postestrus following injections bf estradiol on days 9 and 10. This effect may have been due to two consecutive days of treatment with estrogen rather than only on day 10. Inhibition of prostaglandin synthesis with indomethacin prevented luteolysis induced with estrogen in cows and ewes (Lewis and Warren, 1974, 1977). Increased uterine content of prostaglandin specific cyclooxygenase occurred during late diestrus (Huslig gt_al,, 1979) and inhibition of synthesis of RNA with actinomycin D prevented induction of luteolysis with estradiol (French and Casidy, 1973). Therefore, induction of luteolysis with estradiol is 19 mediated at least in part by increased uterine synthesis of PGF. Progesterone and Luteolysis Administration of progesterone during metestrus generally shortens estrous cycles in cows and ewes (Woody and Ginther, 1968). Hysterectomy prevents this effect of progesterone (Woody gt 31., 1967). Administration of progesterone during metestrus also facilitates estradiol-induced luteal regression on days 4 or 5 postestrus (Warren gt_al,, 1973). Mechanism of Action of PGan The concept that prostaglandins are involved in luteal regression is based primarily on: 1. Exogenous PGan induces luteal regression in a manner similar to that observed histologically in spontaneous regression (McClellan gt al,, 1977). 2. Inhibition of synthesis of prostaglandin prohibits spontaneous luteal regressing (Lewis and Warren, 1976, 1977). 3. Elevated concentrations of PGFZa in utero-ovarian venous blood are temporally associated with luteal regressiOn (Land _e_t_a_l_., 1976; Thorburn _e_t__a_l., 1972; Shemesh and Hansel, 1975a). The mechanism by which PGan exerts its luteolytic effect is still being investigated. There is no evidence that reduced concentrations of gonadotropins in blood are involved in spontaneous or PGan induced luteal regression. Mechanisms still being investigated include reduction 20 in blood flow to CL, interference with binding and/or actions of gonado- trophin, activation of 1ytic enzymes and/or direct effect on luteal metabolism. Blood Flow Pharriss and Wyngarden (1969), aware of the venoconstrictive pro- perties of prostaglandin an and its abundance in uterine secretions. demonstrated exogenous PGan to be luteolytic. Further studies have not been able to provide direct evidence that luteal regression is a result of diminished blood flow to luteal tissue. The ovary bearing a CL has greater blood flow than the contralateral ovary. Moreover, the CL receives the major portion of ovarian blood flow (Niswender gt__a_l_., 1975; Brown _e_t_a_1_., 1980; Bruce and Moor, 1975). Einer—Jensen and McCracken (1976) reported that secretion of progesterone declined prior to reduced capillary blood flow through the CL. Rates of flow for blood through an ovary bearing a corpus luteum decreased only after production of progesterone declined in serum of ewes during luteolysis induced with PGFZa. However, when PGFZa was infused in larger amounts into ovarian arteries, rates of flow decreased immediately by 50 percent (McCracken gt 31,, 1972). Brown gt_aj, (1980) reported a linear relationship between blood velocity in the artery supplying an ovary with a corpus luteum, plasma concentrations of progesterone and luteal weights. Velocity of blood in arteries supplying ovaries without corpora lutea did not change throughout ovine estrous cycles. In pseudopregnant rats, luteal blood flow did not change during 12 hour test period following admfinistration of PGan. Flow of blood to ovarian interstitium increased 21 during this period and did not return to basal flow until after concentrations of progesterone in serum declined fully (Pang and Behrman, 1981). Redistribu- tion of blood flow from luteal tissue to ovarian interstitium and follicles was also observed in rabbits following treatment with PGFZa (Navy and Cook, 1973). Redistribution of blood flow could create an anoxic environment for luteal tissue and thus account for early morphological signs of luteal regres- sion (Deane §t_gl,, 1966). Therefore diminished blood flow to luteal tissue has not been established as a cause or effect of luteal regression. Role of Lysosomes in Luteal Regression Dingle $3.31, (1968) observed increased lysosomal activity in luteal tissue taken from ewes at the time of luteal regression. McClellan gt;al, (1977) reported lysosomes increased in enzyme activity and packaging within luteal cells at the onset of spontaneous and PGFZa induced luteal regression in ewes. Discrete receptors for PGFZa have been detected in lysosomal membranes within bovine luteal cells (Rao gt 31,, 1978). Receptors for LH, PGE, and PGan weredetected on lysosomal membranes and plasma membranes of bovine corpora luteal (Rao gt_al,, 1978; Mitra and Rao, 1978). Affinity of bovine luteal receptors for PGFZa increased at the time of luteal regression prior to alterationsin binding of LH (Rao gt_al,, 1979). Although lysosomes may play a role in degradation of cellular components during luteolysis, it is not known if lysosomes alter steroidogenesis during initial phases of luteal regression. Effects of PGan on Metabolism in Luteal Cells -Secretion of progesterone from luteal tissue incubated in vitro is reduced after 4 hours of exposure to PGan (Demers gt 31., 1973; Henderson 22 and McNatty, 1975; Evrard-Herouard et al., 1981). Marsh (1971) observed an additive effect of PGE2 and LH on luteal synthesis of cAMP and proges- terone 1g_vitro. Henderson and McNatty (1975) suggested that PGE2 increased synthesis of progesterone through increased production of cAMP. Conversely, PGan acted on the coupling component of the LH induced adenylate cyclase system, reducing cAMP and thus decreased synthesis of progesterone. Fitz 33 al. (1980) reported decreased luteal adenylate cyclase activity assoc- iated with decreased concentrations of progesterone in serum. Diekman gt al, (1978b) quantified receptors for LH in luteal pre- parations of ewes following treatment in yiyg_with PGFZa. No change in binding sites for LH were observed until after concentrations of proges- terone in serum had declined. Similarly, characteristics of receptors for LH in bovine luteal tissue were not altered until after spontaneous = luteal regression was near completion (Rao _t__l,, 1979). In summary, alterations in binding of gonadotropic to luteal tissue are not involved in initiation of luteolysis. However, PGan may exert its effect in part by disrupting the adenylate cyclase system without affecting binding of LH. Although lysosomal activity increases during luteal regression and receptors for PGFZa are aSSOciated with lysosomal .membranes, a causative role of lysosomes in luteolysis has not been established. Role of Development of Follicles in Luteal Regression Two major roles of follicles are the production of oocytes and secretion of steroids (Richards, 1978). In view of the effects of estrogen on luteal function, characterization of follicular function throughout the 23 estrous cycle is warranted. During estrous cycles of heifers the pro- portion of atretic (degenerating) follicles to normal follicles remained constant within size category. Most (2/3) follicles were less than 3 mm in diameter in all stages of an estrous cycle. Follicles greater than 4 mm diameter comprised less than 11 percent of the total population of follicles. The number of non-atretic follicles varied greatly among heifers and no cyclic pattern was observed. However, the pattern of growth and atresia of the largest follicle demonstrated a tendency toward two waves of follicular growth during bovine estrous cycles (Rajakowski, 1960). Waves or peaks in follicular activity have also been described in ewes (Brand and deJong, 1973). Matton.gt_al, (1981) reported enhanced ability of bovine ovaries to develop large follicles after day 15 postestrus compared to metestrus or early to mihdiestrus. Numbers of follicles less than 3 mm decreased from days 3 through day 13 postestrus whereas numbers of medium follicles (3 to 6 mm) increased on day 13 and 18 postestrus relative to days 3 or 8. Numbers of large follicles (>6 mm) did not vary among days tested (days 3, 8, 13 and 18 postestrus). A Choudary §t_al, (1968) reported no pattern of growth or atresia to occur during bovine estrous cycles and concluded that growth of follicles from one size class to another is continuous and independent of stage of cycle. Richards (1980) proposed a continuum of follicular growth and atresia occurs between surge of gonadotrophic at proestrus. Marion gt al, (1968) theorized that all follicles that pass through a "competent" stage without estrous stimulation become atretic and are replaced by smaller developing follicles. Follicles can become atretic at any stage of growth. The follicle destined to ovulate does not emerge as the largest 24 follicle until day 18 of the bovine estrous cycle (Dufour t 1., 1973). Moor _t_al, (1978) reported the greatest concentration of estrogen in follicular fluid and greatest secretion of estrogen was associated with large non-atretic follicles. England gt al. (1973) reported the largest follicle on the ovary bearing a CL contained more total estrogen and greater concentration of estrogen than the largest follicle from non—CL ovaries on day 14 of the bovine estrous cycle. However, the size of the largest follicle from ovary bearing CL and non-CL ovary do not differ at this time of the.bovine estrous cycle (Matton et.gl,, 1981). In ewes, presence of a CL has been shown to enhance follicular development (Dufour £31., 1971;Fogwe11e_t__a_1_.,1977). Luteal Function in the Absence of Ovarian Follicles Karsch gt a1. (1970) first demonstrated that arresting follicular development by irradiation lengthened luteal function in ewes. Other studies in ewes confirmed these results (Hixon gt_al,, 1975; Gengenbach et_a_l_., 1977). Additionally, Hixon _e_3_a_1_. (1975) reported exogenous PGan to be less effective in causing luteal regression in ewes with follices destroyed. Administration of estradiol benzoate or estradiol benzoate plus PGan resulted in luteal regression in ewes with or without ovarian follicles. In a follow up experiment a larger dose of PGan was effective in inducing luteolysis in ewes with or without follicles present. However, in hysterectomized ewes, estradiol benzoate with PGan caused luteal regression with follicles present and reduction of progesterone in serum in the absence of follicles (Gengenbach et_al,, 1977). This was the first evidence in ewes that estrogen could facilitate luteal 25 regression in the absence of the uterus. Importantly, estradiol may affect luteal regression from a site in addition to the uterus. Luteal life span has been reported to be increased in cows with visible follicles destroyed by cautery during late diestrus (Chupin and Saumande, 1979). Administration of PGan or estradiol valerate reduced luteal lifespan and interval to estrus in cows with follicles destroyed by cautery (Chupin and Saumande, 1981). Definitive studies addressing the role of ovarian follicles in luteal regression in cows have not been conducted. However, based primarily on studies in sheep, it would appear that ovarian follicles contribute to the process of luteal regression. Effects of X-irradiation on Ovarian Tissue Numerous studies utilizing selective X-irradiation of ovaries have been conducted. Sublethal doses of X-rays cause selective changes in ovarian functions. Cells undergoing division are very sensitive to X-rays during metaphase and during the part of interphase involving synthesis of DNA. While large doses of X-rays can kill a cell, lower doses can affect nuclear chromatin sufficiently to "kill the nucleus" yet cytoplasmic metabolism continues (Baker and Neal, 1977). The inherent lifespan of cells contributes to the metabolic mani- festations of sublethal doses of X-rays on a tissue. Thus, the effects of disruption of cellular division might not detectably alter function of a tissue for a long period (Lacassagne et_al,, 1962). X-irradiation of adrenal tissue resulted in disrupted steroidogenesis over a ten week period (Berliner gt al., 1964). I Corpora lutea were able to develop after X-irradiation of graafian 26 follicles in rabbits (Lacassagne _t“al., 1962). Whole ovary X-irradiation of ewes resulted in occasional incidence of follicles becoming luteinized and/or cystic (Gengenbach eta_1_., 1977; Hixon etal. ,' 1975). In ewes (Hixon gt al., 1975; Karsch gt_gl,, 1970), in monkeys (Hobson and Baker, 1979) and in hamsters (Norman and Greenwald, 1971) whole ovary X-irradia- tion did not impair secretion of progesterone. Whole ovary irradiation at midcycle in ewes resulted in nearly complete absence of follicles, complete impairment of follicular develop- ment and no histological evidence of altered corpora lutea (Karsch gt al., 1970; Hixon gt_al,, 1975; Gengenbach §t_al,, 1977). However, a low incidence of follicular cysts which had luteinized was reported (Hixon et al., 1975; Gengenbach et.al,, 1977). In summary, based on the studies utilizing whole ovary X-irradiation, sublethal irnfdiation of ovaries containing fully functional corpora lutea effectively inhibits follicular development without impariing luteal development. Electrocautery of macroscopically visible follicles removes larger antral follicles but does not appear to inhibit further follicular development. Summary of Review of Literature 1. Corpora lutea influence estrous cycle length. 2. Luteal lifespan is determined by variation in luteolytic factors, not by luteotropic factors. 3. The uterus is an important component in luteal regression. 4. '0varian follicles and estrogens apparently contribute to luteal regression in part via the uterus. . 5. Follicles and presumably estrogens are temporally associated with luteal regression. 27 This study is intended to address the following question. Is the presence of ovarian follicles after midcycle important to the luteolytic process in cows? Furthermore, by monitoring secretory patterns of LH, progesterone, estradiol and PGFZa elucidation of the roles of these hormones in the luteolytic process is possible. MATERIALS AND METHODS General This study was conducted from Fall 1978 through Winter 1979. At midcycle (days 10 or 11 postestrus), cows underwent surgery marking the beginning of the experimental period which ended on day 22 postestrus. In treated cows (X-irradiated, n a 6) follicles were destroyed by X-irradiation and cautery. Controls (shameirradiated, n = 6) were manipulated similarly, but follicles were not destroyed. All cows received indwelling catheters into a jugular vein and both utero-ovarian veins in order to monitor hormonal patterns during the experimental period. Ovaries were removed on day 22 postestrus and returned to the laboratory for inspection and to weigh corpora lutea. Animals ° Non-lactating, parous Jersey and Guernsey cows were observed for estrus 3 times daily. After two or more estrous cycles of 19 to 24 days, cows underwent surgery at midcycle (day 10 postestrus n = 9. day 11 postestrus n = 2). Surgery Feed and water were withheld for 36 hours prior to surgery. Poly- vinyl chloride (PVC) cannulae were installed and secured into a jugular vein to facilitate sampling of blood prior to and throughout the experi- mental period. 28 29 Anesthesia was induced with rapid intravenous administration of a 5% solution of glyceryl guaiacolate (Glycodex, Burns-Bioted Lab, Chromalloy Pharmaceuticals, Oakland, CA) and 0.2% thiamylal (Surital, Parke-Davis, Detroit, MI) to effect.. Upon intubation, anesthesia was maintained with halothane (Fluothane, Ayerst Lab, New York, NY) by inhalation. Cows were transferred to a custom built surgical cradle modeled after that described by Anderson gt al. (1962) and maintained in dorsal recumbency. Following routine preparation of the abdomen, a midventral celiotomy was performed. The incision began at the pubis and extended 25 to 30 cm anteriorly.' The reproductive tract was exteriorized and a uterine branch of both utero-ovarian veins was identified within the mesometrium. A 1 to.2 cm incision was made in mesometrium and tissue dissected down to the vein. After adequate isolation of the vein, it was grasped with vascular forceps and incised transversely. Sterilized PVC cannulae (Ico Rally, Palo Alto, CA) coated with 0.7 TDMAC-heparan comlex (Tri-docecylmethyl ammonium chloride, Polysciences, Inc. Warrington, PA) was placed into each vein and positioned to collect blood from the utero- ovarian vein prior to the vena cava. Cannulae were secured to the mesometrium, marked to identify left and right, filled with heparinized saline (400 u/ml, Ha-heparin US Biochemical Corp., Cleveland, OH) and exteriorized through the paralumbar fossa. Treatment Cows in the X-irradiated group had all follicles at the surface of each ovary destroyed by electro-cautery. For radiation, each ovary was isolated above a 1/8 inch thick lead shield to protect surrounding tissues. Ovaries were irradiated with a General Electric Maxitron - 300 X-ray machine 3O operated at 300 peak kilovolts (300 KvP) and 20 millamperes (20 Ma). Quality of the X-ray beam was 2.0 mm Cu half-value layer. Each ovary received a total dose of 1500 rads over a ten minute interval with a target distance of 50 cm. Ovaries of,sham-irradiated (control) cows were exteriorized and held above the lead shield for 10 to 12 minutes and no follicles were cauterized. Following routine surgical closure, cows were moved to tie stalls for the remainder of each cow's experimental period. Each cow received 6,000,000 units of procaine penicillineG (300,000 u/ml, Pfizer, Inc., New York, NY) initially then 3,000,000 units twice daily for three days. To preserve patency of utero-ovarian venous cannulae, sterilized saline (0.9% NaCl) with heparian (100 ulml) and penicillin-G (Na-penicillin- 0, Sigma Chemical Corp, St. Louis, MO, 1,000 u/ml) was infused continuously through each cannula at the rate of 10 ml/hour with a constant infusion apparatus (Harvard Apparatus Co., Cambridge, MA) for the duration of the experiment or until patency of cannulae was lost. Sampling of Blood Five jugular venous samples were taken 15 minutes apart to character- ize patterns of secretion of LH one hour prior to anesthesia and three times daily for three days after treatment. These samples were to provide control data on LH within and between groups of cows after treatment. Collectively, the five samples will be referred to as LH windows. LH windows were resumed three times daily on days 16 to 19 postestrus in order to determine potential differences in the secretion of LH between treatment groups during the period of anticipated luteal regression. Jugular venous and utero-ovarian venous samples were taken every four hours for the duration of the experiment. Additional samples were 31 taken every hour for eight hours, once daily, on days 15 to 19 postestrus in order to characterize acute changes in the secretion of estradiol-178 and PGan during the period of luteal regression. Each sample from each utero-ovarian vein was.divided between two tubes. Blood in the tube marked for determination of PGan was acidified with 0.1 m1 of 0.1N HCl per ml blood to prevent synthesis of prostaglandins by platelets (Pexton at al., 1975b) Blood in the other tube was reserved for the determination of estradiol-17B. All samples were heparinized upon collection and stored in a refrigerator until centrifugation. Following separation by centri- fugation, plasma was transferred to a new tube, labelled and stored frozen until assayed. Quantification of Hormones Plasma collected via jugular cannulae was reserved primarily for determination by radioimmunoassay of concentrations of LH (Convey et 31,, 1976) and progesterone (Louis gt_al,, 1973). Concentrations of estradiol- 178 were determined in plasma from blood samples taken from utero-ovarian veins throughout the eXperimental period and from jugular venous blood sampled on day 22 postestrus. Assay for estradiol was a modified version of that described by Oxender et_al, (1977). Steroids were extracted from plasma with ether and the extract chromatographed with Sephadex LH—20 (Pharmacia Fine Chemicals, Piscataway, NJ) as described by Butcher et_al, (1974). To account for procedural losses, 5,900 cpm of 3H-1,2,5,7-estradiol- 178 were added to four additional plasma samples in each assay. These samples were extracted and chromatographed with unknowns and recovery was calculated as percentage of total radioactivity recovered. The interassay coefficient of variation was 10 percent. Plasma samples determined to 32 contain more than 25 pg/ml estradiol were reassayed for verification. Because of the specificity of the antibody and chromatographic procedure, results are expressed as pg of estradiol-17B per m1 of plasma. PGon Concentrations of PGFZa were determined in utero—ovarian venous plasma using the extraction procedure described by Pexton et_al, (1975) and chromatography described by Lewis et a1. (1978). This procedure effectively separates PGFla from PGan thus making the assay specific for PGan (Cornette at al., 1972; Stellflug at al., 1975). Results are expressed as ng PGan per ml plasma. Percent recovery was determined in a manner similar to that for estradiol. Ability of the assay to measure 5 ng/ml, 10 ng/ml and 25 ng/ml was demonstrated. However, the coefficient of variation at 0.4 ng/ml was 32 percent. Therefore, surges in secretion of PGan (values greater than 1 ng/ml) can be characterized but sensitivity was not sufficient to measure differences in basal concentrations of PGan (values less than 1 ng/ml). Samples determined to contain more than 1 ng/ml of PGFZa were reassayed to increase confidence in values above ‘basal concentrations. Ovariectomy At the end of the experimental period (day 22 postestrus n = 10, day 20 postestrus n = 2) ovaries were removed from cows through a supra- vaginal incision. Corpora lutea were enucleated from ovaries and following removal of extraneous tissue, weighed to the nearest 0.01 g. Visible follicles were counted and their diameters measured. Ovaries from the X-irradiated group were sliced every 2 mm and inspected visually for 33 antral follicles. Presence of any visible follicles in ovaries which were cauterized and X-irradiated excluded that animal from the experiment. Statistical Analysis. Differences in weights of corpora lutea between treatment groups were evaluated by students t test. Profiles of concentrations of proges- terone were examined for parallelism between groups by least squares regression analysis using time as split plot. Differences between treat- ment groups in mean concentrations of estradiol-17B in utero-ovarian venous plasma on days 10 and 15 and in jugular venous plasma on day 22 postestrus were evaluated by students t test. Orthogonal contrasts were utilized to determine variations in mean concentrations of estradiol-17B in uteno-ovarian venous plasma among days in control animals. Students t test was utilized to determine differences in mean concentrations of LH between treatment groups on each day. RESULTS Efficacy of Treatment 1) No evidence of follicles in ovaries of treated cow at the time of ovariectomy. 2) Cows were verified to be in luteal phase of estrous cycle at time of treatment. This assesment based on inspection of luteal tissue at time of surgery and concentrations of progesterone in blood. Criteria for inclusion of animals in results included: 0f the six cows treated at midcycle with cautery of visible follicles and whole ovary X-irradiation, five had no visible follicles at the time of ovariectomy. One cow had two follicles (14 mm and 8 mm) present in one ovary. Data from this animal were not included in the results. Another treated cow had experienced luteal regression prior to treatment. This assessment was based on concentrations of progesterone in blood samples ' taken prior to surgery. Therefore these results are based on four treated and six control cows. 1 Ovaries of four cows which were X-irradiated following cautery of visible follicles at midcycle had no visible follicles (< 2 mm) on day 22 postestrus. Prior to cautery of follicles and X-irradiation of ovaries concentratibns of estradiol-178 in utero-ovarian venous plasma (Table 1) did not differ between groups. However, on day 15 postestrus cows in the X-irradiated group had lower concentrations of estradiol-178 in utero- ovarian venous plasma compared to the sham-irradiated group (P<.025) or compared to pretreatment levels (P<.005) (Table 1). Figure 1 illustrates 34 35 TABLE 1. CONCENTRATIONS 0F ESTRADIOL-l78 IN PLASMA AND INVENTORY 0F FOLLICLES PRIOR TO AND FOLLOWING X-IRRADIATION OR SHAN-IRRAOIATION.a Treatment n f Estradiol-17B (pg/m1) Number of follicles - ~+ - 3_8 mm per ovary UterO-ovarian Juguflar Pre-treatb Day 15 Day 22 Day 10 Day 22' Sham-irrad. 6 12.9:2.6 16.3:5.8 5.2:].5 0.5:,2 1.5:,4 X-irrad. 4 11.1:fl.5 5.6:0.7* 2.6:0.3**‘ 0.6:,2 0.0 aValues given as mean i_standard error. bBlood samples taken prior to electro-cautery of follicles or sham-cautery. * Less than respective control value (P<0.025). **Less than respective control value (P<0.05). 36 Figure 1. Effect of sham-irradiation or X-irradiation of ovaries on concentrations of estradiol-17B in plasma from utero-ovarian veins draining both ovaries. Values represent daily mean concentrations :_s.e. of all animals within treatment group. / signifies time of treatment. a S'I'RA 6101.47, (ngmI) 37 1- .———-O Shani-irradiated A----A X-irradfatod 25-- ’ ..- . 20-- 15" 10'- 5-1- “\eo-"" ’f----- e---t---i 1 I l I l 1 1'0 1‘1 1‘2 1'3 1'4_ 1 5 DAYS 90813873113 38 the daily mean concentrations of estradiol-178 in utero—ovarian venous plasma draining both ovaries of treated and control cows. Following destruction of follicles, lowest-concentrations of estradiol—17B were Observed after two days following treatment (P<.05). Concentrations of estradiol-17B also declined in plasma from control cows over the same interval (P<.005) however, this trend reversed markedly after day 12. Days 12 to 15 postestrus, concentrations of estradiol-178 in X-irradiated cows remained low with very little variation, while concentrations of estradiol-178 in sham-irradiated cows increased. Concentrations of estradiol-178 in jugular venous plasma on day 22 postestrus (Table 1) were lower in treated cows than in controls (P<.05). Estradiol-178 measured in serum from an ovariectomized cow was 2.2 i_.38 pg/ml which did not differ (Students t test, P>.40) from concentrations of estradiol-178 in jugular venous plasma in cows with no visible ovarian follicles on day 22 postestrus. . Based on these parameters, (reduction in secretion of estradiol-17B and absence of follicles) it is concluded that the combination of elec- trocautery and X—irradiation of ovarian follicles was effective in reducing follicular function and preventing follicular development. Effects of Treatment on Concentrations of LH in Plasma Mean concentrations of LH (Figure 2) in plasma did not differ between groups prior to treatment, within or between treatment groups on any day after treatment. The schedule for sampling was not adequate to examine secretory patterns of LH. It is therefore possible that differences in the pulsatile patterns of LH existed even though basal secretion of LH was not affected by treatment or stages of estrous cycle tested. 39 Figure 2. Effect of cautery of follicles and X-irradiation or sham- irradiation on concentrations of LH in jugular venous plasma. Values represent daily mean concentrations 1 s.e. + signifies time of treatment. 4o N up ‘D n— . . ...u..p.o~ a... Op n— . n - — 3p . u m. a. .. - 133:2..l-an I -b I 1‘? a £3. ("I“) I ‘1 b.— 41 TABLE 2. PLASMA PROGESTERONE AND WEIGHTS OF CORPORA LUTEA OF COWS ON DAY 22 POSTESTRUS. FOLLOWING X-IRRADIATION ON DAY 10. Treatment n Progesterone (ng/ml) Luteal weight (g) Sham 5 2.0 1 ..5a 3.8 2‘. ,.5° X-irradiation 3 4.7 1 1.Ob 8.3 + 1.7d Values presented are mean :_standard error. Values within a column with different superscripts are significantly different (ab, P<.01; Cd, P<.OOl). 42 Effect of Treatment on Luteal Function BaSed on comparisons Of concentrations of progesterone in plasma of control and treated cows, there was no effect of X-irradiation on luteal function through day 20 postestrus. However, by day 22 postestrus mean concentrations of prOgesterone.in plasma (Table 2) of sham-irradiated cows ‘were lower than in X-irradiated cows (P<.01). In addition the profile (Figure 3) of concentrations of progesterone in plasma was maintained in treated cows but declined in control cows (P<.001). Luteal regression had not begun by day 22 postestrus in one control cow. All cows in the treated group had sustained luteal function through day 22 postestrus. Weights of corpora lutea removed from treated cows on day 22 postestrus were greater (P<.001) than those from control cows (Table 2). Based on these results, it is concluded that destruction of follicles prolOnged luteal function. Relationship of secretion of estradiol-178 to progesterone Concentrations of estradiol-17B were determined in utero-ovarian venous plasma of five cows from day 10 postestrus through day 19 to determine if increased secretion of estradiol is associated with luteal regression. Profiles of estradiol-178 and.progesterone are depicted in Figure 4. Based on analysis of variance, secretion of estradiol-178 increases after mid- cycle, prior to decline in progesterone (P<.001). The increase in estradiol- 17B secretion is transient since by day 19 postestrus concentrations of estradiol-17B were not different from those observed at midcycle. Since estradiol-17B increased prior to decline in progesterone, these observa- tions support the notion that estradiol, a product of follicular steroido- genesis, may mediate the effect of follicles in luteal regression. 43 Figure 3. Concentrations of progesterone in jugular venous plasma of cows following electrocautery of follicles, X-irradiation of ovaries or sham-irradiation. 44 PROGESTERONE (ng/ml) A I 01 1 OJ 1 l .N XI _mw>0_>._.mo 1.11 m1>zlsm>o§mo a. _ b _ _w _m _N O><0 «w .n aa a. a. A. - a. a. va «a a. p. o— " a I. a a w u u u u u a u“ % niacin/4‘ 23 j ’4 \ul .2/ u .1: No .a ~f 1 . 1- .-\~. s - a a .: a a tax-«- ans - a ’N .I p I a .f- e I‘. 0 \ as ‘r C . , A J. 11"“) p31“ a uoausaaou ‘[ C l in 1'” DISCUSSION Electra-cautery of surface follicles.and whole ovary X-irradiation was intended to destroy visible follicles and prevent further follicular development. Based on lower concentrations of estradiol-17a on days 15 and 22 postestrus and absence Of visible follicles at ovariectomy on day 22 postestrus this basic goal was achieved. Studies utilizing isolated whole ovary X-irradiation in cattle have not been reported. Studies utilizing follicular cautery in cattle have been reported by Staigmiller egg. (1980) and Chupin and Saumande (1979). In ewes (Dufour _etalu 1971) and in cows (Chupin and Saumande, 1979), electrocautery of follicles delayed but did not prevent onset of estrus. Hixon gt_al, (1975) and Gengenbach gt 21, (1977) reported inhibition of follicular development with whole ovary X-irradiation but luteinization of follicles occurred in some ewes. Lacassagne gt_al, (1962) reported luteinization of follicles following X-irradiation of rabbit ovaries. Luteinization was thought to have occurred in follicles present on the surface of ovaries at the time of irradiation. During development of techniques for this study, luteinized tissue was observed at a site where a follicle existed at the time of cautery and X-irradiation six days previous. It was concluded that cautery of follicles had to be complete and care was taken that minimal follicular tissue remained following electro-cautery. In the present study, luteini- zation of follicular tissue was not observed on any ovary removed on day 22 postestrus following electro-cautery and X-irradiation on day 10 post- estrus. Apparently extensive electro-cautery of surface follicles 54 55 prevented luteinization of follicular tissue. Because large non-atretic follicles are thought to be the primary sources of estradiol (Moor gt_al,, 1978), it was expected that concentra- tions of estradiol-178* in utero-ovarian venous plasma would decline rapidly following electrO-cautery and X-irradiation of ovaries, however the decline occurred over two days. Choudary gt al. (1968) reported all follicles greater than 5 mm diameter (medium and large follicles) present during the luteal phase Of cattle are atretic. Rajakowski (1960) reported most large follicles present at midcycle in heifers are atretic. In the present study there was no apparent relationship between presence of large (>6 mm) follicles on individual ovaries at the time of surgery and concentrations of estradiol-173 at midcycle (days 10 to 12 postestrus) were lower than 'between days 14 and 18 postestrus. Therefore, a likely source of estradiol (secreted at midcycle would be small follicles (<2 mm) which might not have been cauterized, yet retained steroidogenic capacity for a period following X-irradiation. Even by day 15 postestrus, five days after treatment, con- centrations Of estradiol-17B in uterO-ovarian venous plasma of treated cows were still greater than concentrations observed in jugular venous plasma on day 22 postestrus. Therefore, it appears that treatment effect- ively reduces but does not prevent secretion of estradiol. This observation is consistent with the goal of disrupting follicular development without cellular destruction. Critical to understanding the role of follicles in luteal regression” was determining whether or not secretion of the primary luteotropin, LH, was affected by treatment. Following initiation of this experiment, Rahe at 31, (1980) reported high magnitude pulses of LH occurring every three to four hours during diestrus. Therefore, the one hour windows of frequent 56 sampling utilized in the present experiment would not suffice to characterize secretory patterns of LH. However, in this study mean concentrations of LH in jugular venous plasma did not differ between treatment.groups on any day. Therefore, continued luteal function in treated cows was not due to increased basal secretion of LH. - Staigmiller gt al. (1980) reported increased concentrations of LH in cows following cautery Of follicles during proestrus. Beck _t_gl, (1976) reported progesterone and estradiol were both necessary to suppress post- castrational increases in concentrations of LH in heifers. However, in those studies the magnitude Of change in concentratiOn of LH due to treat- ment was large enough to be detected with infrequent sampling of blood. Therefore, in the present study, the failure of secretion of LH to increase following follicular destruction may be due to continued presence of estradiol. Comparison Of concentrations of progesterone in blood of X-irradiated and sham-irradiated cows showed no detectable acute effects of irradiation on luteal function. This is in agreement with studies in ewes utilizing similar doses of X-irradiation (Hixon gt 31,, 1975; Gengenbach et al., 1977). Ichikawa gt a1, (1968) used a higher dose of X-irradiation (2000 rads) on ovaries of ewes at midcycle without altering secretion of proges- terone. Thus, based on unaltered profiles of progesterone after X-irradia- tion in the present study, an additional goal of irradiating ovaries without directly affecting luteal function was realized. The primary objective of this study was to determine whether or not ovarian follicles, present after mid-cycle, affect luteal regression in cows. Based on greater luteal weights and greater concentrations of pro- gesterone in plasma of cows with follicles destroyed than in controls, it is concluded the ovarian follicles are involved in the normal luteolytic 57 process. This effect was also Observed in ewes (Karsch gt_al,, 1970; Hixon e_t__al., 1975; Gengenbach gt_a_1_., 1977) in which follicles were destroyed by X-irradiation. Administration of LH during diestrus can prolong luteal function (Donaldson and Hansel, 1975a). However, Karsch .gtual. (1970) reported a six fold increase in circulating concentration of LH was required to extend luteal lifespan in ewes. Thus, in order for increased secretion of LH to prevent luteal regression large (easily detect- able) increases may be required. The absence of obvious changes in secre- tion of LH in the present study precludes the pituitary as being involved in this process. I I A potential role of follicles in luteal regression is increased secretion of estradiol during a period of luteal and/or uterine sensitivity to the luteolytic effects of estradiol. Secretion of estradiol-178 in control cows as relatively stable at midcycle but significant increases in secretion were observed after day 13 postestrus. This is the first con- clusive evidence that secretion.of estradiol increases prior to the onset of luteal regression in cattle. Concentrations of estradiol-17B were determined in plasma from both utero-ovarian veins in each cow from days 10 through 15 postestrus. Interestingly, with cows in which comparisons could be made, secretory peaks of estrafiOl-l78 (>25 pg/ml) were observed only in samples taken from veins draining ovaries containing a CL. TheSe comparisons, although not definitive, are consistent with observations by England et_al, (1973) that greater concentrations of estradiol are found in fluid from large follicles ‘ on ovaries bearing CL on day 14 postestrus in heifers than from contrala- teral ovaries. Additionally, Dufour et_al, (1971) proposed that corpora lutea promote follicular development via a local (unilateral) mechanism 58 in ewes. Karsch gt a1_(l970) demonstrated a potential local effect of follicles on luteal function in ewes and Fogwell 25.91: (1978) observed a local, yet extraovarian pathway for the transfer of a luteolytic sub- stance (PGan) from follicles to corpora lutea in ewes. Evidence in the present study that secretion of estradiol-17B from the ovary bearing a CL increases.after midcyCle, but prior to luteal regression, illustrates a temporal and local association between follicular steroidogenesis and luteal regression. ' The duration and magnitude of observed peaks of estradiol-178 in utero-ovarian'venous plasma may be masked in peripheral blood. Most studies have failed to detect increased concentrations of estradiol in jugular venous blood just prior to luteal regression. Thus, it would appear that if enhanced secretion of estradiol is involved in luteal regression, it does so Via a local pathway in order to achieve higher concentrations of estradiol in responsive ovarian or uterine tissues. This pathway may be intraovarian or utero-ovarian. Responsive tissues include ovary and uterus. Cook gt al, (1974) demonstrated that intra- luteal injection of estrogen induced luteal regression in ewes. This effect ‘could not be reversed with administration of LH systemically thus further implicating estrogen as being luteolytic locally. The duration of increased secretion of estradiol prior to luteal regression may be important to luteolysis. 391t.§£”21o (1978) reported smaller doses of estradiol were luteolytic if administered over 24 hours versus larger doses over 12 hours. Although in the present study observed peaks in SecretiOn of estradiol-178 were transient, lasting less than two hours, the interval in which peaks occurred lasted more than 24 hours. 59 Thus the average concentration of estradiol-17B in utero-ovarian venous plasma was in fact elevated for a period greater than 24 hours. Interestingly, increased secretion of estradiol observed just prior to luteal regression was probably not associated with follicles destined tO ovulate. In cattle, follicles destined to ovulate are not thought to emerge as the largeSt follicle until day 18 postestrus, 3 to 4 days prior to estrus (Dufour et_al,, 1973). In the present study, four of five cows in the control group ovariectomized on day 22 postestrus had the largest follicle present on the ovary not bearing a CL. Whereas, increased secretion of estradiol-17B was observed in utero-ovarian venous plasma associated with ovaries bearing corpora lutea. Additionally, in cows in which a corpus luteum regressed, concentrations of estradiol-17B in uterO-ovarian venous plasma were increased only transiently, having returned to basal concentrations by day 22 postestrus. These observations do not preclude the possibility that follicles influencing luteal regression are from a pool of follicles from which the ovulatory follicle emerges (Brand and deJong, 1973). Yet a discrete increase in secretion of estradiol occurs prior to luteal regression and does not contribute directly to proestrous secretory patterns of estradiol-17B. Importantly, this increase in secretion Of estradiol may be crucial and unique to initiation of luteal regression. Characterization of acute changes in secretion and temporal associa- tions of PGFZa, estradiol-178 and progesterone in individual control cows during diestrus may help pinpoint potential roles of these hormones in the luteolytic process. Secretory patterns of estradiol-17B and PGan were characterized in two cows prior to and during luteal regression and in another cow which had not initiated luteal regression by day 22 6O postestrus. In the cows undergoing luteolysis, increased secretion of estradiol-178 was followed by increased secretion of PGan prior to decreases in concentrations of progesterone. In the cow with prolonged luteal function no increases in estradiol-17B or PGan were Observed in utero-ovarian venous plasma nor were any follicles greater than 5 mm Observed on ovaries removed on day 22 postestrus. These observations are consistent with the theory that ovarian follicles influence luteal regression by increased secretion of estradiol which directly and/or indirectly by increased synthesis and release of PGFZa cause luteal regression. Lewis and Warren (1977, 1974) implicated synthesis of prostaglandin by the uterus as a mediator of estrogen induced luteolysis in cattle and ewes. While most studies implicate the uterus as necessary for estrogen induced or spontaneous luteolysis, other studies indicate an intra- ovarian element of luteal regressiOn may also be involved. Shemesh and Hansel (1975b) demonstrated synthesis of PGF from follicles and luteal tissue in vitro. In monkeys, exogenous estradiol induces luteolysis, at least in part through increased ovarian synthesis of prostaglandins (Auletta letflal., 1978). In ewes, uptake of estradiol into CL changes during diestrus (Sheridan at al., 1975) and intra-luteal injections Of estrogen caused luteolysis (Cook 3111., 1974). Binding proteins for estrogen are reported to exist in bovine 1uteal.tissue (Kimball and Hansel, 1974). In hysterectomized ewes, administration of estradiol benzoate reduced the dose of PGan necessary to induce luteal regression (Gengenbach et_al,, 1977). Although one should consider both ovarian and uterine contributions of PGan in the luteolytic process, other studies gave evidence for luteal regression induced by estradiol independant of prostaglandin an. 61 Gengenbach gt_al, (1977) reported 3.5 mg PGan to be luteolytic if given with estradiol benzoate to hysterectomized ewes with or without follicles present. Seven mg PGan was luteolytic in four of four ewes with follicles present but luteolytic in only two of four ewes with follicles destroyed by X-irradiation. In cows with large (>4 mm) follicles destroyed by electrocautery, time from cautery to luteolysis was shortest if PGFZa was given at the time of cautery or if given two days after treatment of cautery plus estradiol valerate. These observations lead one to consider estrogen and PGan act synergistically in the luteolytic process. However, one should not rule out the possibility that the effect is merely additive with estrogen's contribution being stimulation of further synthesis of prostaglandin. Although peaks in secretion of estradiol-17B and PGan were observed in utero-ovarian venous plasma prior to decreased production of proges- terone, no direct temporal associatiOn was apparent among these hormones. Robinson gt 31, (1976) found removal of endometrial tissue, a primary source of uterine PGFZa resulted in greater concentrations of progesterone in blood of ewes during diestrus. In the present study, concentrations of progesterone in plasma were not different between control and treated cows until luteal regression began in controls. Moreover, variation in concentrations of progesterone in plasma was observed on all days in all cows independent of treatment. In the,present study, concentrations of PGan in utero-ovarian venous plasma of sham-irradiated cows did not vary from basal levels until late diestrus when significant elevations were observed. However, during this period variations in concentration of progesterone in jugular venous plaSma occurred. Thus variations in concen- trations of progesterone observed during diestrus are not always associated 62 with increased release of PGan into utero-ovarian venous blood. Addition- ally, absence of significant changes in secretion of estradiol-173 between days 10 and 13 postestrus preclude estradiol-178 as a source of variation in luteal function during this period. Although-not tested in the present study, it would be of interest to examine the temporal association between secretory patterns Of LH and progesterone. Interpretation of data involving concentrations of progesterone in blood determined by radioimmunoassay is potentially confounded by the possibility that variations are in part due to storage and processing of blood samples (Vahdat at al., 1981). One cow in the control group failed to undergo luteal regression by day 22 postestrus. ~At ovariectomy (day 22 postestrus) no follicle greater than 5 mm in diameter were present on either ovary. No signif- icant changes in Secretion of estradiol-17B or PGan were detected in utero-ovarian venous plasma from this cow. It would appear that failure Of luteolysis was due to absence of follicles, no increase in secretion of estradiol and no increase in secretion of PGFZa. The informatiOn learned from this study along with that reviewed can be synthesized into a model for the timing of luteal regression (Figure 8). Because the antiluteolytic effect of an embryo is not evident until mid to late diestrus, a period of luteal function, independent of conception is necessary for the reproductive process. Although secretion of estrogen and PGFZo may be increased during metestrus, during early development (Period A) corpora lutea are resistant to the luteolytic influences of PGan and estradiol. During days 5 to 8 postestrus (B) estradiol is not luteolytic due to its inability to stimulate synthesis of PGan. Exogenous PGFZa is luteolytic during this period. Estradiol attains its luteolytic ability after day 8 postestrus (B and C) due to a period of progestational 63 influence. However days 8 to 14 (C) is a period of follicular estrogenic quiescence. After day 14 (D) postestrus, follicular steroidogenesis increases and secretion of estradiol is increased for 3 to 4 days. This extended (>24 hr) period of estrogenic influence is necessary for luteolysis. The luteolytic effeCt of estradiol is mediated at least in part through increased synthesis and release of prostaglandin. Significant increases in concentrations of PGan in uterO-ovarian venous plasma follow a period of increased secretion of estradiol. In summary, luteal regression in cattle is initiated by increased steroidogenesis Of ovarian follicles present after midcycle. Increased secretion of PGFZa into utero-ovarian venous blood occurs after the increased secretion of estradiol and is followed by a decline in secretion of progesterone (luteolysis). The question remains as to what initiated follicular steroidogenesis observed to increase during late diestrus. 64 Figure 8. Hormones that influence luteal regression in cattle: A model of the bovine estrous cycle. 65 O 0 #1 A I s r 11.- . H In .>‘C - D p pr )O-N 0.9 MPPI Nun fi-p mun ’F ”n— N-—. PPP Obp 9. G. W @- O- V p- p l1. 1‘ “In.“ .flOIHIUIHIB "I”.“ullzuquI-O" \ /a/ \ 0 . ~ o \\ . a o o o o o 111 11.. fl Kuloc.uc0.ufl.ooul to o 1 a o ‘2 LO. 9.....- ocouozounzl 00"T. LIST OF REFERENCES LIST OF REFERENCES Akbar, A.H., L.E. Reichert, Jr., T.G. Dunn, C.C. Kaltenbach and G.D. Niswender. 1974. Serum levels of follicle-stimulating hormone during the bovine estrous cycle. J. Anim. Sci. 39:360. Anderson, L.L., A.M. Bowerman and R.M. Melampy. 1965. Oxytocin on ovarian function in cycling and hysterectomized heifers. J. Anim. Sci. 24:964. Anderson, L.L., F.C. Neal and R.M. Melampy. 1962. Hysterectomy and ovarina function in beef heifers. J. Vet. Res. 23:974. Asdell, S.A., J. deAlba and S.J. Roberts. 1949. Studies on the estrous cycle of dairy cattle: cycle length, size of corpus luteum, and endometrial changes. Nornell Vet. 39:389. Auletta, F.J., H. Agins and A. Scommegna. 1978. Prostaglandin F mediation of the inhibitory effect of estrogen on the corpus luteum of the Rhesus monkey. Endocrinology 103:1183. Baker, T.G. and O. Neal. 1977. Action of ionizing radiations on the mammalian ovary. In The Ovary Vol. 111. L. Zuckerman and B.J. Weir (Ed.) Academic Press. New York. Barcikowski, B., J.C. Carlson, L. Wilson and J.A. McCracken. 1974. The effect of endogenous and exogenous estradiol-173 on the release of prostaglandin an from the ovine uterus. Endocrinology 95:1340. Beck, T.W. and E.M. Convey. 1977. Estradiol control of serum luteinizing hormone concentrations in the bovine. J. Anim. Sci. 45:1096. Beck, T.W., V.G. Smith, B.E. Sequin and E.M. Convey. 1976. Bovine serum LH, GH and prolactin following chronic implantation of ovarian steroids and subsequent ovariectomy. J. Anim. Sci. 42:461. Behrman, H.R., A.K. Hall, S.C. Preston and 5.0. Gore. 1982. Antagonistic interactions of adenosine and prostaglandin F a modulate acute responses of luteal cells to luteinizing ho ne. Endocrinol. 110:38. . 66 67 Berliner, D.L., W. Stevens and M.L. Berliner. 1964. The effects of ionizing radiation on the biosynthesis and biotransformation of corticosteroids. In: Effects of Ionizing Radiation on the Re roductive S stem. W.D. Carlson and F.X. GassnerTEd.) MacMillan Co., New York. Black, D.L. and R.T. Duby. 1965. Effect of oxytocin, epinephrine and atropine on the estrous cycle of the cow. J. Reprod. Fert. 9:3. Bolt, D.J. and H.W. Hawk. 1972. Failure of esogenous estrogens to induce CL regression in hysterectomized ewes. J. Anim. Sci. 35:237 (Abstr.). Bolt, D.J. 1973. Reduced luteolytic effect of PGFZa by hysterectomy or hCG in ewes. J. Anim. Sci. 34:302 (Abstr.). Bolt, D.J. 1978. The influence of dose and duration-of estrogen treatment on corpus luteum regression in ewes. Theriogenology 9:3 3. Brand, A. and W.H.R. deJong. 1973. Qualitative and quantitative micromorphological investigations of the tertiary follicle pOpulation during the oestrous cycle in sheep. J. Reprod. Fert. 33.431. Brown, B.W., M.J. Emery and P.E. Mattner. 1980. Ovarian arterial blood velocity measured with doppler ultrasonic transducers in conscious ewes. J. Reprod. Fert. 58:295. Bruce, N.W. and R.M. Moor. 1975. Ovarian follicular blood flow in the sheep. J. Reprod. Fertil. 43:392. Brunner, M.A., L.E. Donaldson and W. Hansel. 1969. Exogenous hormones and luteal function in hysterectomized and intact heifers. J. Dairy Sci. 52:1849. Butcher, R.L., W.E. Collins and N.W. Fugo. 1974. Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17B throughout the 4-day estrous cycle of the rat. Endocrinology 94:1704. Butler, H.R., D.J. Bolt and P.V. Malven. 1971. Transient surges of LH in ovariectomized sheep. The Physiologist 14:117 (Abstr.). Charkraborty, P.K. and F. Stormshak. 1976. Role of the uterus in oestradiol induced luteal regression in the ewe. J. Reprod. Fertil. 48:431. Chenault, J.R., N.W. Thatcher, P.S. Kalra, R.M. Abrams and C.J. Wilcox. 1975. Transitory changes in plasma progesterone, estradiol and luteinizing hormone approaching ovulation in the bovine. J. Dairy Sci. 38:509. 68 Choudary, J.B., H.T. Gier and G.B. Marion. 1968. .Cyclic changes in bovine vesicular follicles. J. Anim. Sci. 27:468. Christian, R.E. and L.E. Casida. 1948. The effects of progesterone ‘ in altering the estrous cycle of the cow. J. Anim. Sci. 7:540 Abstr. . Chupin, D. and Saumande, J. 1979. New attempts to decrease the variability of ovarian response to PMSG in cattle. Ann. Biol. Anim. Biochem. Biophys. 19:1489. ~ Chupin, D. and J. Saumande. 1981. Effect of exogenous prostaglandin and/or estrogen on luteolysis after electrocauterization of the largest follicles at the end of the bovine estrous cycle. Therio. Enol. 16:497. Condon, W.A. and J.L. Pate. 1981. Influence of serum and its lipoprotein fractions on progesterone synthesis and secretion by bovine luteal tissue jn_vitro. Biol. Reprod. 25:950. Convey, E.M., W.E. Beal, B.E. Sequin, K.J. Tannen and Y.C. Lin. 1976. Gonadotropin releasing hormone induced luteinizing hormone release after prostaglandin an in heifers. Proc. Soc. Exp. Biol. Med. 151:84. Cook, 8., F.J. Karsch, D.L. Foster and A.V. Nalbandov. 1974. Estrogen induced luteolysis in the ewe: Possible sites of action. Endocrino- logy 94:1197. Cornette, J.C., K.T. Kirton, K.L. Barr and A.D. Forbes. 1972. Radio- immunoassay of prostaglandin an. J. Reprod. Med. 9:355. Deane, H.W., M.F. Hay, R.M. Moor, R.M. Rowson and R.V. Short. 1966. The corpus luteum of the sheep: Relationships between morphology and function during the oestrous cycle. Acta. Endocrinol. 51:245. DelCampo, C.H. and O.J. Ginther. 1973. Vascular anatomy Of the uterus and ovaries and the unilateral luteolytic effect of the uterus: Horses. sheep and swine. Am. J. Vet. Res. 34:395. Demers, L.M., H.R. Behrman and R.0. Greep. 1973. Effects of prostaglan- dins and gonadotropins on luteal prostaglandin and steroid bio- synthesis. Adv. Biosci. 9:701. Denamur, R., J. Martinet and R.V. Short. 1973. Pituitary control of the ovine corpus luteum. J. Reprod. Fertil. 32:207. Desjardens, C. and H.D. Hafs. 1968., Levels of pituitary FSH and LH in heifers from birth through puberty. J. Anim. Sci. 27:472. Dona Dufc Dui Du 69 Diekman, M.A., P. O'Callaghan, T.M. Nett and 6.0. Niswender. 1978a. Validation of methods and quantification of luteal receptors for LH throughout the estrous cycle and early pregnancy in ewes. Biol. Reprod. 19:999. Diekman, M.A., P. O'Callaghan, T.M. Nett and 6.0. Niswender. 1978b. Effect of prostaglandin F a on the number of LH receptors in ovine corpora lutea. .Biol. RepFod. 19:1010. Dingle, J.T., M.F. Hay and R.M. Moor. 1968. Lysosomal function in the corpus luteum of the sheep. J. Endocrinol. 40:325. Dobson, H. 1978. Radioimmunoassay of FSH in the plasma of postpartum dairy cows. J. Reprod. Fertil. 52:45. Dobson, H. and P.D.G. Dean. 1974. RadiOimmunoassay of oestrone, oestradiol-178 and 17a in bovine plasma during the oestrus cycle and last stages of pregnancy. J. Endocrinol. 61:479. Donaldson, L. and W. Hansel. 1965. Histological study of bovine corpora lutea. J. Dairy Sci- 48:905. Dufour, J., O.J. Ginther and L.E. Casida. 1971. Corpus luteum action ' on ovarian follicular development after destruction of macrosco- pically visible follicles in ewes. Proc. Soc. Exp. Biol. Med. 138:475. Dufour, J., H.L. Whitmore. O.J. Ginther and L.E. Casida. 1973. Identification of the ovulating follicle by its size on different days of the estrous cycle in heifers. J. Anim. Sci. 34:85. Dutt, R.H. and L.E. Casida. 1948. Alteration of the estrual cycle in sheep by use of progesterone and its effect upon subsequent ovulation and fertility. Endocrinology 43:208. Einer-Jensen, N. and J.A. McCracken. 1976. Krypton measurement Of capillary blood flow in the ovine corpus luteum during PGF a induced luteolysis. .In: Recent Advances in Prostaglandin 5nd Thgomboxane Research Vol. 2, . . 90 . England, B.G., H.J. Karavolus, E.R. Hauser and L.E. Casida. 1973. Ovarian follicular estrogens in Angus heifers. J. Anim. Sci. 37:1176. ‘ Erb, R.E., R.D. Randel and C.J. Callahan. 1971. Female sex steroid changes during the reproductive cycle. J. Anim. Sci. Suppl. 1. 32:80. . amuelsson and R. Paoletti 70 Evrard-Herouard, M., M.P. delaLlosa-Hermier, J. Martinet, P. Mauleon and C. Hermier. 1981. LH receptors in ovine corpora lutea in relation to various physiological states and effects Of PGan on LH induced steroidogenesis ig.vitro. J. Reprod. Fertil. 61.225. Fitz, T.A., J.L. Fleeger, MOF. Smith.and P.G. Harms. 1980. Human chorionic gonadotrOpin (hCG) binding and adenylate cyclase (AC) activity in deVeloping and regressing corpora jutea (CL). Biol. Reprod. 22: Suppl. 1:61A (Abstr.). Fogwell, R.L., 5.5. Lewis, R.L. Butcher and E.K. Inskeep. 1977. Effects of ovarian bisection on response to intrafollicular injection of PGFZa and on follicular development in ewes. J. Anim. Sci. 45:328. Fogwell, R.L., C.H. Weems, C.S. Lewis, R.L. Butcher, and E.K. Inskeep. '> 1978. Secretion of steroids after induced luteal regression in beef heifers: Effects of PGon and removal of corpora lutea. J. Anim. Sci. 46:1718. Ford, S.P., C.H. Weems, R.E. Pitts, J.E. Pexton, R.L. Butcher, and E.K. Inskeep. 1975.' Effects of estradiol-17B and progesterone on prostaglandins F in sheep uteri and uterine venous plasma. J. Anim. Sci. 41:1407. French, L.R. and L.E. Casida. 1973. Effect of actionomycin D on corpus luteum regression in ewes. J. Anim. Sci. 37:1218. Barverick, H.A., R.E. Erb, G.D. Niswender and C.J. Callahan. 1971. Reproductive steroids in the bovine: III Changes during the estrous cycle. J. Anim. Sci. 32:946. Gemmell, R.T., B.D. Stacey, and 6.0. Thorburn. 1974. Ultrastructural study of secretory granules.in the corpus luteum of the sheep during the estrous cycle. Biol! Reprod.-11:447. Gemmell, R.T., B.D. Stacey and G.D. Thorburn. 1976. Morphology of the regressing corpus luteum in the ewe. Biol. Reprod. 14:270. Gengenbach,.D.R., J.E. Hison and w. Hansel. 1977. Aluteolytic inter- action between estradiol and prostaglandin an in hysterectomized ewes. Biol. Reprod. 16:571. Ginther, O.J., C.0. Woody, S. Mahajan, K. Janakiraman and L.E. Casida. 1967. Effect of.oxytocin administration on the oestrus cycle of -uni1aterally hysterectomized heifers. J. Reprod. Fert. 14:225. Ginther, O.J. 1967. Local utero-ovarian relationships. J. Anim. Sci. 26:578.. Ginther, O.J. 1970. Length of estrous cycle in sheep treated with estradiol. Am. J. Vet. Res. 31:973. 71 Ginther, O.J; and C.H. DelCampo. 1974. Vascular anatomy of the uterus and ovaries and the unilateral luteolytic effect of the uterus: Cattle.. Am. J. Vet. Res. 35:193. Glencorss, R.G., I.B. Munro, B.E. Senior and C.S. PopeL' 1973. Con- centrations Of oestradiol-17B, oestrone and progesterone in jugular venous plasma offcows during the estrous cycle and in early pregnancy. Acta. Endocrinol. 73:374. Goodman, R.L., S.M. Pickover and F.J. Karsch. 1981. Ovarian feedback control of follicle-stimulating hormone in.the ewe. Evidence for selective suppression. Endocrinology 108:772. Greenstein, J.S., R.H. Murray and R.C. Folly. 1958. Effect of exogenous hormones on the reproductive rocesses of the cycling dairy heifer. J. Dairy Sci. 41:1834 (Abstr. . Hackett, A.J. and H.D. Hafs. 1969. Pituitary and hypothalamic endocrine changes during the bovine estrous cycle. J. Anim. Sci. 28:531. Hansel, W. and W.C. Wagner. 1960. Luteal inhibition in the bovine as a result Of oxytocin injections, uterine dilation and intrauterine infusions of seminal and prepUtial fluids. J. Dairy Sci. 43:796. Hansel, W. and S.E. Ecternkamp. 1972. Control of ovarian function in domestic animals. Amer. 2001. 12:225. Hansel, W., P.W. Concannon and J.H. Lukaszewska. 1973. Corpora lutea of the large domestic animals. Biol. Reprod. 8:222. Hansel, W., J. Hixon, M. Shemesh and D. Tobey.. 1976. Concentrations and activities of_prostaglandins Of the F series in bovine tissue, blood and milk. Symposium: Prostaglandins in Bovine Reproduction. Hauger, R.L., F.J. Karsch and 0. Foster. .1977. A new concept for control of the estrous cycle of the ewe based on the temporal relatiOnship.between LH, E2 and P in peripheral serum and evidence t3?t88;ogesterone inhibits tonic LH secretion. 'Endocrinology Henderson, K.M. and K.P..McNatty.' 1975. A biochemical hypothesis to explain the mechanism of luteal regression. Prostaglandins 9:779. Herriman, I.D., D.J. Harwood, C.B. Mallinson.and R.J. Heitzman. 1979. ‘ Plasma concentrations of ovarian hormones during the estrous cycle of the sheep and cow. J. Endocrinol. 81:61. Hixon, J.E. and M.T. Clegg. 1969. .Influenceof thepituitary on ovarian progesterone output in the ewe:- Effects of hypophysectomy and gonadotrophic hormones. Endocrinology 84:828. 72 Hixon, J.E. and W. Hansel. 1974. Evidence for preterential transfer of prostaglandin F a to the ovarian artery following intrauterine administration in Zatt1e. Biol. Reprod. 11:543. Hixon, J.E., O.R. Gengenbach and W. Hansel.~ 1975. Failure of prostar glandin F a to cause luteal regression in ewes after destruction of ovariafi follitles by X-irradiation. Biol. Reprod. 13:126. Hobson, W.C. and W. Hansel. 1972. Plasma LH levels after ovariectomy, corpus luteum removal and estradiol administration in cattle. EndocrinolOgy 91:185. Hobson, B.M. and T.G. Baker. 1979. Reproductive capacity of Rhesus monkeys following bilateral ovarian X-irradiation. J. Repro. Fertil. 55:471. Horton, E.W. and N.L. Poyser. 1976. Uterine luteolytic hormone: A physiological role for prostaglandin an. Physiol. Rev. 56:595. Howland, B.E., W.M. Palmer and L.M. Sanford. 1978.. Changes in serum _1evels of LH and FSH in progesterone-treated ovariectomized ewes following a single injection of estrogen. Can. J. Anim. Sci. 58:15. Huslig, R.L., R.L. Fogwell and W.L. Smith. 1979. The prostaglandin forming cyclooxygenase of ovine uterus: Relationship to luteal fUnction. Biol. Reprod. 21:589. Ichikawa, Y.,-J.A. McCracken, D.T. Baird and A. Uno. 1968. Effect Of'X-ray irradiation on ovarian steroid secretion in the sheep. Proc. Int. Cong. Endocrinol. (Abstr. 210):84. "Inskeep. E.K., W.J. Smutney, R.L. Butcher and J.E. Pexton. 1975. Effects of intrafollicular injections of prostaglandins in non- pregnant and pregnant ewes. J. Anim. Sci. 41:1098. Kaltenbach, c.c., C.D. Niswender, D.R..Simmerman and J.H. Wiltbank. ‘> 1964. Alteration of ovarian activity in cycling, pregnant and hysterectomized heifers with exogenous estrogens. J. Anim. Sci. 23:995. . ' Kaltenbach, C.C., 8. Cook, G.D. Niswender and A.V. Nalbandov. 1967. Effect Of pituitary hormones on progesterone synthesis by oviae luteal tissue in_vitro. Endocrinology 81:1407. Kaltenbach, C.C., J.W. Graber, 6-0. Niswender and A.V. Nalbandov. 1968. Luteotrophic properties of some pituitary hormones in non-pregnant or pregnant ewes. ~Endocrinology 82:818. 73 Karsch,'F.J., J. W. NovecOSke,'J. F. Roche, H. W. Horton and A. V. Nalbandov. 1970. Maintenance of ovine corpora lutea in the absence of ovarian follicles. Endocrinology 87: 1228. Karsch, F. J. smf G. P. Sutton. 1976. An intra-ovarian site of the luteolytic action of estrogen in the Rhesus monkey. Endocrinology . 98: 553. Karsch, F.J., D.L. Foster, S.J. Legan, K.D. Ryan and G.K. Peter. 1979. A'Control of the preovulatory endocrine events in the ewe: Interrela- .tionship of estradiol, progesterone and luteinizing hormone. Endocrinology 105:421. Kesner, J.K. 1981. ”Control of the preovulatory luteinizing hormone and follicle stimulating hormone surges in cattle.‘ Ph.D. Dissertation, ‘ Michigan State University. Keyes, P.L. and A.V. Nalbandov. 1967. Maintenance and function of corpora lutea in rabbits depend on estrogen. .Endocrinology 80:938. Kimball, F.A. and W..Hanse1.‘ 1974. EstrOgen binding proteins in bovine endometrium and corpus luteum. Biol. Reprod. 11:566. Lacassagne, A., J.F. Dupian, H. Harcovich and A. Raynaud. 1962. The action of ionizing radiations on the mammalian ovary. In: The_ Ovary Vol. II. S. Zuckerman (Ed.) Academic Press, New York. Land, R.B., D.T. Baird and R. J. Scaramuzzi. ‘ 1976. Dynamic studies of prostaglandin F a in the utero-ovarian circulation of the sheep. J. Reprod. Fertil. 47: 209. Lewis, C.S., P.E. Jenkins, R.L. Fogwell and E.K. Inskeep. 1978. Concentrations of prostaglandins E and F a and their relationships to luteal function in early pregna t ewes. J. Anim. Sci. 47:1314. Lewis, P.E. and J.E. Warren, Jr. 1977. Effect of indomethacin on estrogen-induced luteolysis in the ewe. Prostaglandins 13:957. Lewis, P.E. and J.E. Warren, Jr. 1976. Intra-uterine infusion of indomethacin and luteal function in Holstein heifers. J. Anim. Sci. 43:293 (Abstr.). Lewis, P.E. and J.E. Warren, Jr. 1974. Indomethacin inhibits estrogen- induced luteolysis in heifers. J. Anim. Sci. 39:993 (Abstr.). Louis, T. H., H. D. Hafs and B. F. Sequin. 1973' Progesterone, LH, estradiol and ovulation after prostaglandin an in hiefers. Proc. Soc. Exp. Biol. Med. 143: 152. -Loy, R.G., R.G. Zimbleman and L.E. Casida. 1960. Effects of injected Ovarian hormones on the corpus luteum of the estrual cycle in cattle. J. Anim. Sci. 19:175. 74 Mapletoft, R.J., H.R. DelCampo and O.J. Ginther. 1975. Unilateral luteotropic effect of uterine venous effluent of a gravid uterine horn in sheep. Proc. Soc. Exp. Biol. Med. 150:129. Marion, 6.8., H.T. Gier and J.B. Choudary. 1968. Micromorphology of the bovine ovarian follicular system. J. Anim. Sci. 27:451. Marsh, J. 1971. The effect of prostaglandins on the adenyl cyclase of the bovine corpus luteum. Ann. N.Y. Acad. Sci. 180:416. Matton, P., V. Adelakoun, Y. Couture and J.J. Dufour. 1981. Growth - and replacement of the bovine ovarian follicles during the estrous — cycle. J. Anim. Sci. 52:813. McClellan, M.C., Jr., J.H..Abel, Jr. and 6.0. Niswender; 1977. Function of lysosomes during luteal regression in normally cycling and PGan- treated ewes. Biol. Reprod. 16:499._ McCracken, J.A., M.E. Glew and~R;J. Scaramuzzi. 1970. Corpus luteum regression induced by prostaglandin an.- J. Clin. Endocrinol. Metab. 30:544. ~ McCracken, J.A. 1971. Prostaglandin an and corpus luteum regression. Ann. N.Y. Acad. Sci. 180:456. McCracken, J.A., J.C. Carlson, H.E. Blew, J.R. Goding and D.T. Baird. 1972. Prostaglandin identified as a luteolytic hormone in sheep. (Nature New Biol. 238:129. MclamPY. R.M. and L.L. Anderson. 1968. Role of the uterus in corpus luteum function. J. Anim. Sci. 27:77 Suppl. I. Mitra, S. and Ch. V. Rao. 1978. Receptors for gonadotropins and prostaglandins in lysomsomes of bovine corpora lutea. Arch. Biochem. Biphys. 185:126. Moeller, A.N. and N.L. VanDemask. 1951. The relationship of the interval between insemination to bovine fertility. J. Anim. Sci. 10:988. Moor, R.M., M.F. Hay, H.M. Dott and 0.6. Cran. 1978. Macroscopic identification and steroidogenic function of atretic follicles in sheep. J. Endocrinol. 77:309. ‘ Nett, T.M., R.B. Staigmiller, A.N. Akbar, M.A. Diekman, W.E. Ellingood and 6.0. Niswender. 1976. Secretion of prostaglandin an in cycling and pregnant ewes. J. Anim. Sci. 42:876. Newcomb, R., W.D. Booth and L.E.A. Rowson. 1977. The effect of oxytocin treatment on the levels of prostaglandin F in the blood of heifers. J. Reprod. Fertil. 49:1724. ' '75 Niswender, G.D., C.C. Kaltenbach, R.P. Shumway, J.N. Wiltbank and D.R. Zimmerman. 1965. Alteration of ovarian activity in cycling beef heifers with samll daily injections of estadiol. J. Anim. Sci. 24:986. Niswender, G.D., R.T. Moore, A.M. Akbar, T.M. Nett and M.A. Diekman. 1975. Flow Of blood to the ovaries of ewes throughout the estrous cycle. Biol. Reprod. 13:381. ' Norman, R.L. and 6.5. Greenwald. 1971. Effect of phenobarbital, hypophysectomy and X-irradiation on preovulatory progesterone levels in the cyclic hamster. Endocrinology 89:598. Novy, M.J. and M.J. Cook. 1973. Redistribution of blood flow by prostaglandin an'in the rabbit ovary. Amer. J. Obstet. Gyn. 117:381. Owens, R.E., M.D. Cullier, D.C. Kramer, J.L. Fleeger and P.G. Harms. 1981. Effect of ovariectomy and progesterone replacement on patterns of plasma luteinizing hormone (LH) in the bovine. J. Anim. Sci. : (Abstr.). Oxender, W.D., P.A. Noden and H.D. Hafs. 1977. Estrus, ovulation and serum progesterone, estradiol and LH concentrations in mares after an increased photoperiod during winter. Am. J. Vet. Res. 38:203. Pang, C.Y. and H.R. Behrman. 1981. Acute effects of prostaglandin an on ovarian and luteal blood flow, luteal gonadotropin uptake in. vivo and gonadotrOpinbinding in vitro. Endocrinology 108:2239. Pexton, J.E., S.P. Ford, L. Wilson, Jr., R.L. Butcher and E.K. Inskeep. 1975. Prostaglandins F in the uterine tissue and venous plasma of ewes with intrauterine devices. J. Anim. Sci. 41:144. Pharriss, 8.8. and L.J. Wyngarden. 1969. The effect of prostaglandin F a on the progestogen content of ovaries from pseudogpregnant r ts. Proc. Soc. Exptl. Biol. Med. 130:92.‘ Piper, E.L. and W.C. Foote.. 1965. A luteotropic effect of estrodiol in the ewe. J. Anim. Sci. 24:920. Quirk, S.J., D.L. Wilcox, D.M. Parry and 6.0. Thorburn. 1979. Subcellular location of progesterone in the bovine corpus luteum: A biochemical, morphological and cytochemical investigation. Biol. Reprod. 20:1133. Rahe, C.H., R.E. Owens, J.L. Fleeger, H.T. Newton and P.G. Harms. 1980. Pattern of plasma luteinizing hormone in the cyclic cow: Dependance upon the period of the cycle. Endocrinology 107:498. Rajakowski, E. 1960. The ovarian fOllicular system in sexually mature heifers with special reference to seasonal, cyclical and left-right variations. Acta:*Endocrinol. Suppl. 52:1. 76 Rao, Ch. V. , V. L. Este rgreen, F. R. Carman, G. E. Moss and K .A. Frandle. 1978. Gonadotropin and prostaglandin (PG) Fa receptors in bovine corpora lutea (CL) of early, mid and late luéeal phase. Fed. Proc. 37:724 (Abstr.). Rao, Ch. V. , N. L. Estergreen, F. R. Carman Jr. , and G. E. Moss. 1979. Receptors for gonadotropin and prostaglandin F a in bovine corpora lutea of early, mid and late luteal phase. Acéa Endocrinol. 91:529. Rao, Ch. V, S. Mitra and F. R. Carman, Jr. 1981. Characterization of gonadotropin binding sites in the intracellular organalles of bovine corpora lutea and comparison with plasma membrane sites. J. Biol. Chem. 256:2628. Richards, J. S. 1980. ‘ Maturation of ovarian follicles: Actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol. Rev. 60: 51. Roberts, J.S. and J.A. McCracken. 1976. Does prostaglandin F a released from the uterus by oxytocin mediate the oxytocic action of oxytocin. Biol. Reprod. 15:457. Robinson, J.S., J.R.G. Challis, B.J.A. Furr, T.M. Louis and 6.0. Thorburn.- 1976.15 the sheep corpus luteum subject to tonic inhibition during the luteal phase of the estrous cycle. Eur. J. Obstet. Gynec. Reprod. Biol. 6: 191. Roche, J.F. 1976. Calving rate of cows following insemination after 12-day treatment with silastic coils impregnated with progesterone. J. Anim. Sci. 43:164. Roche, J.F. and J:J. Ireland. 1981. The differential effect of pro- gesterone on concentrations of luteinizing hormone and follicle stimulating hormone in heifers. Endocrinology 108:568. Sawyer, H.R., J.H. Abel, Jr., M.C. McClellan, M. Schmitz and 6.0. Niswender. 1979. Secretory granules and progesterone secretion by ovine corpora lutea jg_vitro. Endocrinology 104:476. Scaramuzzi, R.J., B.V. Caldwell and R.H. Moor. 1970. Radioimmunoassay of LH and estrogen during the estrous cycle of the ewe. Biol. Reprod. 3:110. Sharma, S.C. and R.J. Fitzpatrick. 1974. Effect of oestradiol-17B and oxytocin treatment on prostaglandin F alpha release in the anoestrous ewe. Prostaglandins 6:97. Sheldrick, E.L., H.D. Mitchell and A.P.F. Flint. 1980. Delayed luteal Egggession in ewes immunized against oxytocin. J. Reprod. Fert. 77 Shemesh, M., N. Ayalon and H.R. Linder. 1972. Oestradiol levels in the peripheral blood of cows during the oestrous cycle. J. Endocrinol. 55:72. Shemesh, M. and W. Hansel. 1975a. Levels of prostaglandin F (PGF) in bovine endometrium, uterine venous, ovarian arterial and jugular plasma during the estrous cycle. Proc. Soc. Exp. Biol. Med. 148:126. . . ' Shemesh, M. And W. Hansel. ~1975b. Stimulation of prostaglandin synthesis . in bovine ovarian tissues by arachidonic acid and luteinizing hormone. Biol. Reprod. 13:448. Sheridan, P.J., J.L. Philips, K.R. Simmons, J.L. Caffrey, J.H. Abel and -' G.D. Niswender. 1975. Modulation of the uptake and retention of estradiol-17B in the ovine corpus luteum by luteinizing hormone. Proc. Soc. Exp, Biol. Med. 149:537. Short, R.E., R.B. Staigmiller, R.D. Randel and R.A. Bellows. 1979. Factors affecting estrogen-induced LH release in the cow. Biol. Reprod. 21:683. Snook, R.B., M.A. Brunner, R.R. Saatman and W. Hansel. 1969. The affect of antisera to bovine LH in hysterectomized and intact heifers. Biol. Reprod. 1:149. Spicer, L.J., J.J. Ireland and J.F. Roche. 1981. Changes in serum LH, ' progesterone and specific binding of lZS-I-hCG to luteal cells during regression and development of bovine corpora lutea. Biol. Reprod. 25:832. Staigmiller, R.B., B.G. England and L.W. Shappa. 1980. Regrowth of bovine follicles after cautery. Biol. Reprod. 22:110A (Abstr.). Stellflug, J.N., T.M. Louis, H.D. Hafs and B.E. Sequin. 1975. ' Luteolysis, estrus and ovulation, and blood prostaglandin F after intramuscular administration of 15, 30 or 60 mg prostaglandin an. Prostaglandins 9:609. Stellflug, J.N., T.M. Louis, R.C. Gorewit, H.D. Oxender and H.D. Hafs. 1977. Luteolysis induced by prostaglandin F a before and after hysterectomy in heifers. Biol. Reprod. 17:535. Stormshak, F., H.E. Kelley and H.W. Hawk. 1969. Suppression of ovine luteal function by 17B-estradiol. J. Anim. Sci. 29:476. Suter, D.E., P.W. Fletcher, P.M. Sluss, L.E. Reichert, Jr. and 6.0. Niswender. 1980. Alterations in the number of ovine luteal receptors for LH and progesterone secretion induced by homologous hormone. Biol. Reprod. 22:205. 78 Thorburn, 6.0. and P.E. Mattner. 1971. Anastomosis Of the utero-ovarian and anterior mammary veins for collection of utero-ovarian venous blood: Progesterone secretion rates in cyclic ewes. J. Endocrinol. 50:307. ' Thorburn, G.D., R.I. Cox, W.B. Currie, B.J. Restall and W. Schneider. 1972. Prostaglandin F concentration in the utero-ovarian venous plasma of the ewe during the oestrus cycle. J. Endocrinol. 53:325. Umo, I. 1975. Effect of prostaglandin an on the ultrastructure and function of sheep corpora lutea. J. Reprod. Fertil. 43:287. Vahdat, F., J.P. Hurtgen, H.L. Whitmore and B.E. Sequin. 1981. Decline in assayable progesterone in bovine plasma: Effect of time, tempera- ture, anticoagulant and presence of blood cells. Am. J. Vet. Res. 42:521. Ward, K.E., L.C. Longwell, J.L. Kreider and R.A. Godke. 1976. Effect of unilateral hysterectomy on cycling beef heifers. J. Anim. Sci. 43:309. . Warren, J.E., H.W. Hawk and D.J. Bolt. 1973. Evidence for progestational priming of estradiol-induced luteal regression in the ewe. Biol. Reprod. 8:435. Wetteman, R.P., H.D. Hafs, L.A. Edgarton and L.V. Swanson. 1972. Estradiol and progesterone in blood serum during the bovine estrous cycle. J. Anim. Sci. 34:1020. Willcox, D.L. and H.R. Alison. 1982. Release of protein which binds progesterone from the bovine corpus luteum. J: Endocrinol. 92:51. Wiltbank, J.N., J.E. Ingalls and N.W. Rowden. 1961. Effects of various forms and levels of estrogens alone or in combination with gonado- tropins on the estrous cycle of beef heifers. J. Anim. Sci. 20:341. Wiltbank, J.N. and L.E. Casida. 1956. Alteration of ovarian activity by hysterectomy. J. Anim. Sci. 15:134. Woody, C.0. and O.J. Ginther. 1968.‘ Effect of exogenous progesterone on corpora lutea in unitaterally hysterectomized heifers. J. Anim. Sci. 27:1387. Woody, C.O., A.L. First and A.L. Pope. 1967. Effect of exogenous progesterone on estrous cycle length. J. Anim. Sci. 26:139.