Effect of Ovariectomy on Pituitary Gonadotropins in the Cow1

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forms of hypocaleemia disorders induced experimentally in dairy cows. Nord. Vet. Med., 8: 507. (3) Engel, R. W., W. A. Hardison, R. 1~. Miller, N. O. Price, and J. T. Huber. 1964. Effect of copper intake on concentration in body tissues and on growth, reproduction and production in dairy cattle. J. Animal Sci., 2 3 : 1160. (4) Fontenot, J. P., R. :~. Miller, and N. O. Price. 1964. Effects of calcium level and zinc

supplementation of fattening lamb rations. J. Animal Sei., 23:874. (5) Price, N. O., and W. W. Moschler. 1965. Effect of residual lime in soil on minor elements in plants. Agr. l~ood Chem., 13 : 163. (6) Stott, G. H. 1965. Parturient paresis related to dietary phosphorus. J. Dairy Sei., 48: 1485. (7) Underwood, E. J. 1962. Trace Elements in Human and Animal Nutrition. 2nd ed. Academic Press, Inc., New York.

Effect of Ovariectomy on Pituitary Gonadotropins in the Cow 1 Abstract To study changes in pituitary £oUiclestimulating and luteinizing hormone contents following ovariectomy, 24 heifers were spayed on the day of estrus and sacrificed on Day 17 or Day 42. Twelve intact heifers were sacrificed on Day 17 of the estrous cycle. Individual anterior pituitary glands from all animals were assayed for follicle-stimulating hormone by biological assay, and for luteinizing hormone by radioimmunoassay. Pituitary glands from ovariectomized heifers did not differ significantly in fresh or dry weight from those of control animals. Folliclestimulating hormone content in pituitary glands from all spayed heifers was higher (P < .001) than in intact heifers, whereas pituitaries from spayed heifers at Day 42 had more (P < .05) follicle-stimulating hormone than did those at Day 17. Luteinizing hormone content for the pituitary glands of the intact heifers was significantly (P < .005) higher than the average for glands from all spayed heifers.

Introduction An increase in gonad-stimulating activity of the anterior pituitary of the rat following 1 This work was done under a cooperative agreement between the Research Division of the College of Agricultural and Life Sciences, University of Wisconsin, and the Dairy Cattle Research Branch, USDA, Beltsville, Maryland. It was supported in part also by The Ford Foundation Grant 63-505 and by the Program Project in Genetics, Grant GM 15422, from the National Institutes of Health. Published with the approval of the Director of the Research Division of the College; Paper 1423 from the Laboratory of Genetics. JOURNAL OF DAIRY SCIENCE VOL. 54, NO. S

ovariectomy was noted as early as 1929 (4). Individual hormones, follicle-stimulating hormone ( ~ S H ) and luteinizing hormone (LH) continue to increase for weeks above the general level in the pituitaries of the nonpregnant rat (2,5,13). Changes in the pig (9) have been similar when ovariectomy was near the middle of the estrous cycle but the levels, while greater than those for the estrous cycle, did not appear to exceed those at Day 25 of pregnancy or post-hysterectomy. Cyclic changes in pituitary gonadotropins and the greater role of the corpus luteum in species with long estrous cycles make it desirable that the studies on the influence of ovariectomy be related to the stage of the estrous cycle when the ovaries are removed and also to the day of the cycle when intact animals are studied. The anterior pituitary gland of the heifer appears to be relatively high in ~ S H and L I t at Day 17 of the estrous cycle (3,10). The following experiment was designed to determine if spaying would increase F S H and LH more than a normal cycle.

Experlmenta| Procedures Thirty-six nulliparous Holstein heifers weighing 280 to 360 kg were in this study. Twentyfour heifers were totally ovariectomized approximately 12 hr after beginning of estrus. Following ovariectomy two groups of 12 heifers each were sacrificed at Day 17 and Day 42. A control group of 12 intact heifers was sham-operated on the day of estrus and sacrificed on Day 17 of the cycle. Ovarieetomy was through an incision in the dorsal wall of the vagina. At autopsy anterior pituitary glands from all heifers were individually-weighed, homogenized and freeze-dried. Follicle-stimulating hormone activity was tested by a modification

T E C H N I C A L NOTES

of the human chorionie gonadotropin (HCG) synergism method (12), two doses (12.5 and 25.0 rag) per pituitary with two rats per dose. Individual doses were packed in Number 5 gelatin capsules and implanted subcutaneously into Holtzman female rats 30 days old. Each rat was injected subcutaneously with HCG (total dose = 25 IU) twice daily for three days, beginning the day of implantation. On the fifth day following capsule implantation, rats were autopsied and both ovaries removed, cleaned, and weighed. The rat ovarian weight was an index of F S H activity. The double antibody radioimmunoassay procedure modeled after that of Midgley (7) estimated LH content in individual pituitary glands. An aliquant of freeze-dried powder from each gland was extracted twice with phosphatebuffered saline at pH 7.5. The two extracts were pooled, and the protein content of each pool was determined by the Lowry method. Protein values were the bases for quantities for the assays. The antiserum, at a dilution of 1:40,000, was raised in a rabbit by injections with ovine LH together with complete Freund's adjuvant. The LH for labeled tracer and for standard was kindly supplied by Dr. Leo Reichert, Jr. (LER 1056-C2). In our own system 1 ng of that preparation was immunologically equivalent to 2.7 ng N I H ovine LH-S13 and to 15 ng NIH bovine LH-B5. Results and Discussion

Information on weight changes of the anterior pituitary gland after spaying of cows

TABLE 1. Anterior pituitary weights and luteinizing hormone content of intact and spayed heifers, a Treatment

Anterior pituitary gland Fresh wt (mg) Dry wt (mg) Luteinizing hormone (ng/pituitary) h

Intact Day 17 of estrous cycle ],140 281 872

607

is limited. Observations on weight changes in spayed rats are contradictory (1). In the present study no significant changes were observed in weights of fresh or dry anterior pituitary glands of heifers following spaying (Table 1). Radioimmunoassay for LH indicated that pituitaries from intact heifers contained more LH than the average for glands from all spayed heifers (872 versus 568 ng, P < .005). Luteinizing hormone contents of pituitary glands removed 17 and 42 days following ovariectomy were not significantly different (Table 1). Although there was a significant overall dose response in test rat ovary weights (end point for F S H activity), there was also a nonparallelism of dose response between treatments (Table 2). The average ovarian weight for five test-rats injected with HCG alone, 55 rag, did not differ significantly from the average ovarian weight of the test rats on the low dose, 72 rag. Consequently, full expression of the dose difference with the less potent glands was prevented and for this reason, data on the low dose were disregarded. Data for the high dose showed that pituitary glands from intact heifers were less potent than the average of those of Day 17 and Day 42 (98 versus 134 rag, P < .001) and also potency of glands from Day 17 heifers was less than that of Day 42 spayed heifers (122 versus 146 mg P < .05). Thus, in the spayed heifers F S H activity increased well beyond the level in the intact heifers at Day 17. The increase in pituitary F S H potency TABLE 2. Dose x treatment interaction for follicle-stimulating hormone assay: average rat ovary weights. Treatment of heifers

Spayed Spayed 17 days 42 days 1,260 303

433

1,]90 237 528

a Means for 12 heifers per group except that pituitary luteinizing hormone content for animals 17 days after spaying was determined for only 11 heifers. b LER 1056-C2, ovine luteinizing hormone, reference standard.

Dose of heifer pituitary gland

Intact Day 17 of estrous cycle

Low (12.5 rag) High (25.0 nag)

(mg) 67 98

Spayed Spayed Day 17 Day 42 (rag) 72 122

(mg) 76 146

Average ovary weight of five rats treated with human chorionic gonadotropin alone = 55 rag. Within group M.S. for low dose = 562 and for high dose = 1,558 when the responses were analyzed separately. Each treatment contained 12 heifers. JOURI~AL OF DAIRY SCIENCE ~7OL. 54, NO. 3

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following ovariectomy appears to follow a trend similar to that in rats (2,5) and pigs (9). I n mice (8) and sheep (6) where data arc available on both p i t u i t a r y and serum F S t t concentrations, a significant increase was recorded following ovarieetomy. On the other hand, in none of the species cited has there been a decrease in L H following ovariectomy as in heifers. I n t e r p r e t a t i o n s of storage or release of gonadotropins following spaying are speculative in the absence of information on F S H and L H in plasma o f cows. H e i f e r s were spayed 12 h r a f t e r the beginning of estrus at which time, presumably, the p i t u i t a r y gland was depleted of gonadotropic activity by recent release. The intact heifers used as controls were sacrificed at D a y 17 of the cycle at which time p i t u i t a r y gonadotropins are presumed to be relatively high. The results provide evidence that F S H is synthesized and stored in the p i t u i t a r y gland up to 42 days following removal of ovaries whereas L H did not show any evidence of further storage at 17 days, and there was even less present at 42 days. This differential change in p i t u i t a r y content of F S H and L H in heifers emphasizes the need f o r a better understanding of feedback relationships between ovarian estrogen and progesterone and p i t u i t a r y FSI-I and L H synthesis, storage, and release. SYED SAIDUDDIN, ROBERT F. ROWE, K. W. THOMPSON, and L. E. CASIDA, Laboratory of Genetics and Department of Gynecology and Obstetrics, The University of Wisconsin, Madison 53706 References (1) Allanson, M., and A. S. Parkes. 1966. In: Marshall's Physiology of Reproduction. 3rd ed. Vol. 3, p. 206. (2) Cozens, D. A., and M. M. Nelson. 1961. Effects of ovarieetomy on the F S H and ICSH content of the anterior pituitary of the rat. Endocrinology, 68: 767. (3) Desjardins, C., and H. D. Hers. 1968. Levels of pituitary F S t t and L H in heifers from

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(4)

(5)

(6)

(7)

(8) (9)

(10)

(11)

(12)

(13)

birth through puberty. J. Animal Sei., 27: 472. Evans, H. M., and M. E. Simpson. 1929. A comparison of anterior hypophyseal implants from normal and gonadeetomized anim a l s with reference to their capacity to stimulate the immature ovary. Amer. J. Physiol., 89 : 371. Labhsetwar, A. P. 1969. Pituitary levels of F S H and LIt at various intervals after ovariectomy in the rat. J. Reprod. Fert., 18: 531. l~cDonald, P. G., and M. T. Clegg. 1966. Some factors affecting gonadotropin levels in sheep. Proc. Soc. Exptl. Biol. Med., 121: 482. Midgley, A. R., Jr. 1966. Radioimmunoassay: A method for human chorionic gonadotropins and human luteinizing hormone. Endocrinology, 79 : 10. Parlow, A. F. 1964. Effect of ovariectomy on pituitary and serum gonadotropins in the mouse. Endocrinology, 74: 102. Parlow, A. F., L. L. Anderson, and R. M. Melampy. 1964. Pituitary follicle stimulating hormone and luteinizing hormone concentrations in relation to reproductive stages of the pig. Endocrinology, 75: 365. Rakha, A. M., and H. A. Robertson. 1965. Changes in levels of follicle stimulating hormone and inteinizing hormone in the bovine pituitary gland at ovulation. J. Endocrinology, 31: 245. Saiduddin, S., J. W. Riesen, W. J. Tyler, and L. E. Casida. 1968. Relation of postpartum interval to pituitary gonadotropins, ovarian follicular development and fertility in dairy cows. Studies on the Postpartum Cow, Res. Bull., 270, p. 215, Research Division, College of Agriculture and Life Sclence, The University of Wisconsin. Steelman, S. L., and F. M. Pohley. 1953. Assay of the follicle stimulating hormone based on the augmentation with human chorionie gonadotropin. Endocrinology, 53 : 604. Yamamoto, M., N. D. Diebe], and E. M. Bogdanove. 1970. Analysis of initial and delayed effects of orchideetomy and ovariectomy on pituitary and serum L H levels in adult and immature rats. Endocrinology, 86: 1102.