Prolongation of the luteal phase by prostaglandin E2 during the estrous cycle in the cow. A preliminary report

THERIOGENOLOGY PROLONGATION OF THE LUTEAL PHASE BY PROSTAGLANDIN E DURING THE ESTROUS CYCLE IN THE COW. A PRELIMINARY REPhRT. T. Gimenezl and D. M Henricks' 2 Departments of Animal Science1 and Food Science Clemson University, Clemson, SC 29631 Received for publication: October 22, 1982 Accepted: February 23, 1983 ABSTRACT An experiment was conducted to determine the effects of prostaglandin E2 (PGE2) on ovarian progesterone secretion during the estrous cycle in the cow. Intraluminal uterine catheters were implanted in three beef cows (2 treated, 1 control), and 1.3 mg of PGE were infused into the 2 uterus every 4 hours from days 9 through 21 post-estrus. Blood samples were collected from the jugular vein at 2-hour intervals from days 9 to 21 and twice daily from day 22 to 28 post-estrus. Progesterone was measured by applying a specific, direct plasma radioimmunoassay in all samples without extraction. Intrauterine infusion with PGE2 resulted in maintenance of luteal-phase progesterone secretion until day 21 post-estrus, 4 days after luteal regression occurred in the vehicletreated cow. In this study, we demonstrated that PGE2 can prolong the presence of luteal phase plasma progesterone concentrations by possibly stimulating -in vivo steroidogenesis by the corpus luteum during the estrous cycle in the cow. INTRODUCTION Inhibition of prostaglandin synthesis with indomethacin during early pregnancy has delayed or prevented implantation in laboratory animals (l), suggesting that prostaglandins play an important role in establishing pregnancy. Furthermore, PGE has stimulated progesterone secretion by the bovine (2,3) and human (2 ,5) corpus luteum in vitro, and intrauterine PGE2 treatment has resulted in prolonged sheep estrous cycles (6). The purpose of this study was to examine the effect of intrauterine treatment with PGE on _in vivo luteal function during the estrous cycle in the cow. 2 MATERIALS AND METHODS Animals and Treatment Three 6 to 8 year-old crossbred beef cows with moderate body condition scores of 5 were used (7). These animals had lost their calves during

The authors wish to thank The Upjohn Company'for supplying the prostaglandin E2 used in this study, and Mrs. Sandra L. Gray for her technical assistance. This paper is published with the approval of the Director as paper No. 2101, South Carolina Agriculture Experiment Station, Clemson, SC 29631. 1 Correspondence should be addressed to T. Gimenez. MAY

1983 VOL. 19 NO. 5

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THERIOGENOLOGY the periparturient period approximately 50 days before the experiment, The animals were restrained by a and had already shown estrus once. pens under a shed and fed 2 kg of cracked halter, kept in individual Animals corn/animal/day, along with bermuda hay and water -____ ad libitum. were allowed to move freely twice daily for approximately 40 min in a They were observed for onset of estrus in the presence of a large pen. During the luteal phase of the cycle, each bull with a deviated penis. cow received 25 mg of prostaglandin F ct (Lutalyse, The Upjohn Company, One day 8 of the Michigan) to synchronize tg e estrous cycle. Kalamazoo, estrous cycle subsequent to the PGF e injection, catheters were he uterus was exposed through a I_ , surgically placed in the animals. mid-ventral incision with the cow under general anesthesia consisting Five cm of a silicone rubber catheter of halothane and succinylcholine. in a caudal direction into (O.D. 1.19 mm, I.D. .64 mm) were introduced the lumen of the horn ipsilateral to the corpus luteum through a small The catheter puncture approximately 5 cm from the utero-tubal junction. was secured with suture to the myometrium and the opposite end exteriorThe dead volume of the catheter ized through the left paralumbar fossa. A 0.2 urn filter (Acrodisc, Gelman Sciences, Inc., Ann was 0.291 ml. and a three-way stopcock were connected to the catheter Arbor, Michigan) which was stored in a plastic pouch sutured to the left lumbar region. The PGE was stored in absolute ethanol at 4'C as recommended by Stehle 7 8) and diluted with PBS daily as needed. PGE dissolved in I$ or at least 6 absolute ethanol will show less than 10% degradation months at 4'C. by thin layer The purity of the PGE2 was determined chromatography and was found to be 99.4% pure (mean of two assays Each individual performed at The Upjohn Company, Kalamazoo, Michigan). in 100 ul absolute ethanol and uterine infusion consisted of 1.3 mg PGE (1 ml) was 900 nil phosphate buffer saline (PBS). T& e above solution infused into the catheter through the filter every 4 hours (0800, 1200, 1600, 2000, 2400 and 0400 hours) from day 9 at 0800 h to day 21 at 0400 1 post-estrus. The same protocol was followed for the control cow except that only vehicle solution was infused into the uterus. An indwelling catheter (14Ga x 5?', Angiocath, Deseret Co., Sandy, Utah) was placed in a jugular vein also on day 8 post-estrus, and 7 ml of blood were collected according to the following schedule: Day

9 post-estrus at 0700, 0730, 0830 h and every until day 21 post-estrus at 0630 h.

Day

21 post-estrus thereafter

at 1700 and twice daily until day B 28 post-estrus.

The blood was placed until assayed.

in ice,

centrifuged

and

2 hours

thereafter

(0800 and 1700

the plasma

h)

stored

at -20'~

Both uterine horns were surgically removed within five days after collecting the last blood sample, and examined macroscopically for location of the catheter and signs of infection.

694

MAY 1983 VOL. 19 NO. 5

THERIOGENOLOGY Progesterone

assay

Plasma progesterone was measured in duplicate using a specific radioimmunoassay as described previously (9) with the following modifications. The plasma was diluted 1:5 with O.lM PBS-Gel (0.1% gelatin, pH 7.0) by The plasma-PBS-Gel was adding 800 pl of PBS-Gel to 200 pl of plasma. Two 100 1.11aliquots of the then mixed in a Vortex mixer for 5 seconds. solution were placed in test tubes together with 100 pl of antibody (titer 1:30,000) and 100 pl (approximately 7000 cpm) of H3-progesterone To validate these (New England Nuclear, Boston, Massachusetts). modifications, progesterone was measured in 39 plasma samples using both The correlation and regression solvent extraction and direct measurement. coefficients obtained after comparing both procedures are shown in Table 1.

Progesterone (ng/ml) Concentration Range

No. of Samples

Correlation coefficient

0.07 - 0.82

17

0.70

0.78

P/O.01

1.0

22

0.86

1.11

P'O.001

39

0.96

1.09

P
- 4.7

all samples

Table

1.

Correlation and regression coefficients concentrations using solvent extraction measurement in plasma.

Regression coefficient

from progesterone or direct

RESULTS The administration of PGE2 in the manner described resulted in the prolongation of the progesterone secretory activity beyond that seen in the untreated animal (Figure 1). The mean daily plasma progesterone concentrations of the three cows ranged between 1.9 and 3.8 ng/ml from day 9 to 16 of the cycle. On days 17 and 18 post-estrus the progesterone concentrations in the control cow decreased from 4.7 ng/ml to 0.3 ng/ml and remained under 1 ng/ml until day 4 after treatment (Figure 2), then increased during the following 3 days. No signs of estrus were observed in this animal during the experiment. Mean daily progesterone concentrations ranging between 2.7 and 5.0 ng/ml were maintained beyond day 17 until the end of the treatment on day 21 post-estrus in both treated cows (Figure 1). The mean daily progesterone concentration fell sharply in cow No. 2 within the following 24 hours and remained below 0.4 ng/ml until the end of the blood collection period (Figure 1). Standing estrus was observed in this cow on day 4 after treatment. In cow No. 3, the mean plasma progesterone concentrations began to decrease gradually within one day after the last PGE infusion and remained below 1 ng/ml from day 3 to 7 after treatment (F&e 1). When looking at the individual samples, however, the progesterone concentration fell from 6.2 to .7 ng/ml 12 hours after the last PGE2 infusion (Figure 2). The

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progesterone concentration then increased to 2.9 ng/ml during the following day and decreased again until day 3 after treatment. A clear mucous discharge from the vagina was observed in this cow on day 3 after treatment. No signs of infection or morphological differences could be found in any of the cows after macroscopically Also, the catheter was observed in the examining both uterine horns. uterine lumen when examined after hysterectomy. DISCUSSION To our knowledge, this is the first report on the ___ in viva effect of PGE The maintenince on progesterone secretion during the bovine estrous cycle. of high progesterone concentrations in plasma beyond the time of normal luteolysis probably results from the steroidogenic influence of PGE at the corpus luteum level as has been previously shown -__ in vitro (2,3,2,5). When PGE was infused in the uterus of ewes every 4 hours from day 9 interval was increased from 17 through 3 5 post-estrus, the inter-estrus to 38 days (6). However, a significantly prolonged estrous cycle was not observed when the infusion began on day 12 in a similar study in sheep (lo), or on day 14 with 4 infusions of PGE2/day in pigs (11). These studies suggest that to prolong the corpus luteum lifespan, the effect of PGE should start well in advance of the expected onset of luteolysis du?ing the estrous cycle. Stimulation of steroidogenesis in vitro by PGE2 in human corpus luteum -__ during the menstrual cycle compares to that produced by hLH or hCG (5). of If this is true in vivo, no difference in the mean concentrations progesterone among the treated and control animals between days 9 and 16 of the cycle in the present study suggests that either the stimulatory effects of LH and PGE on progesterone production are not additive, or PGE did not stimulatg LH release as shown in the bull after IM injection Although no signs of estrus were observed in the control cow (12% post-treatment, the progesterone concentrations rose above 1 ng/ml plasma on day 3 after treatment indicating that ovulation occurred some Evidence of high estrogen time between days 19 and 24 post-estrus. activity also was shown in the treated cows on days 3 and 4 post-treatment by a clear mucous discharge from the vagina in one cow and standing estrus in the other. PGE, is capable of stimulating steroidogenesis by the corpus luteum during the estrous cycle; however, there is conflicting evidence concerning its role on ovarian function during early pregnancy. PGE2 is synthesized by the sheep embryo and endometrium (13), the bovine blastocyst (14) and early pregnant rat uterus (1). However, no observed difference in the uterine plasma concentrations of PGE between pregnant and non-pregnant sheep on days 15 and 16 post-estrus (1 5) weakens the premise that the PGE, produced by the uterus and embryo is being released to maintain the corpus luteum. Furthermore, -__ in vitro experiments have shown that PGE PGFt;)andIzGA may 1' also stimulate steroidogenesis by the bovine corpus luteurn addition to the steroidogenetic effect of PGE on the ovary after uterine 2 infusion PGE might also have some LH-releasing effect as is the case when administered2directly to the hypothalamus in the rat (16). The corpus luteum of the estrous cycle and that of pregnancy seemed to respond

MAY 1983 VOL. 19 NO. 5

697

progesterone

CYCLE

to the end of the

2. Plasma

FIGURE

of the cycle

OF ESTROUS

DAY

blood

COW

*

DAYS

collection

concentrations

I/ \

COW

*

period.

day

AFTER

from

2, TREATED

1, CONTROL

17

TREATMENT

A!!

estrus

No. 3, TREATED

No.

No.

standing

COW

0

THERIOGENOLOGY differently in the presence of high concentrations of PGE2, since the administration of PGE2 during late pregnancy result in abortion or premature parturition in the cow (17) and human (18). Due to the limited number of animals used in this study, the results obtained are only preliminary and their validity will be further tested with a larger number of cows. The findings in the present study were based on a practical improvement in We found that direct measurement of the assay of progesterone in plasma. progesterone concentrations in plasma was more accurate and precise than As can be seen from Table 1, the accuracy of the extraction procedure. increased once the levels exceeded 1 ng/ml plasma. the direct measurement REFERENCES 1.

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Marsh, J. M. The stimulatory effect cyclase in the bovine corpus luteum,

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stimulation of in Speroff, L. and Ramwell, P. W. Prostaglandin vitro progesterone synthesis. J. Clin. Endocr. 30:345-350 (1970).

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Maeyama, M., Saita, K., Ichihara, K., Munemura, M. and Mori, N. Effect of prostaglandin E2 on adenosine-3', 5'-monophosphate accumulation and progesterone synthesis in human corpora lutea in vitro. J. Steroid. Biochem. 1:295-300 (1976). --

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Marsh, J. M. and LeMaire, W. J. Cyclic AMP accumulation and steroidogenesis in the human corpora luteum: Effect of gonadotropins and prostaglandins, J. Clin. Endocrinol. Metab. x:99-106 (1974).

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Reynolds, L., Huie, J., Stalcup, B., Magness, R., Hoyer, G. and Weems, C. Effect of PGEl or PGE2 on CL in ewes. J. Anim. Sci. -51:Suppl. 1:321 (1980).

7.

Spitzer, J. C. Productivity of range cows of varying body condition, Proc. 2nd Ann. Int. Livestock Symp. (1982) pp. llA-11F.

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Stehle, R. The physical chemistry, stability and handling of prostaglandins E2, F2 alpha, D2 and Dl: A critical summary, in Methods in Enzymology W. E. M. Lands & W. L. Smith (Ed) Academic Press -86:436-458 (198;).

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Rawlings, N. C., Kennedy, S. C., Chang, C. H., Hill, J. R. and Henricks, D. M. Onset of seasonal anestrus in the ewe. J. Anim. Sci. -44:791-797 (1977).

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of prostaglandin E2 on adenylFEBS Letters 1:283-286 (1970).

Pratt, B. R., Butcher, R. L. and Inskeep, E. K. Antiluteolytic effect of the conceptus and of PGE in ewes, J. Anim. Sci. -46: 2 784-791 (1977).

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Schneider, T. M., Tilton, J. E., Okrasa, S., Jeng, M., Weigl, R. and Williams. G. L. Effect of intra-uterine infusions of prostaglandin E (PGE2) on luteal function in non-pregnant gilts, Am. Sot. Anim. Bci. Ann. Meet. Abstr. No. 62 (1982).

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Haynes, N. B., Collier, of prostaglandin E and and prolactin in bills.

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Hyland, J. l-i., Manns, J. G. and Humphrey, W. D. Prostaglandin production by ovine embryos and endometrium in vitro, J. Reprod. Fert. -65:299-304 (1982).

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Shemesh, M., Milaguir, I?.,Ayalon, N. and Hansel, W. Steroidogenesis and prostaglandin synthesis by cultured bovine blastocysts, J. Reprod. Fert. -56:181-185 (1979).

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Lewis, G. S., Jenkins, P. E., Fogwell, R. L. and Inskeep, E. K. Concentrations of prostaglandin E2 and F2" and their relationship to luteal function in early pregnant ewes, J. Anim. Sci. -47:13141323 (1978).

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Ojeda, S. R., Jameson, H. E. and McCann, S. M. Hypothalamic areas involved in prostaglandin (PG)-induced gonadotropin release. I: Effects of PGE and PGF a implants on luteinizing hormone release, Endocrinolo& *:&S-1594 (1977).

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Zerobin, K., Jiichle,W., and Steingruber, Ch. Termination of pregnancy with prostaglandin E (PGE ) and F2n (PGF2a) in cattle, Prostaglandins &:891-961 (1.933).

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Karim, S. M. M. The induction of abortion with prostaglandins. Research in Prostaglandins L:l-3 (1971).

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