In vitro progesterone synthesis by corpora lutea induced in prepuberal cattle

IN

VITRO LUTEA

PROGESTERONE SYNTHESIS INDUCED IN PREPUBERAL

BY CORPORA C A T T L E 1, 2

C. H. SPILMAN, 3 G. E. SEIDEL, JR., 4 L. L. LARSON AND R. H. FOOTE Cornell University, Ithaca, New York 14850 the original demonstration that S INCE slices of porcine corpora lutea (CL) synthesize progesterone in vitro (Duncan et al., 1960), this technique has been used extensively to study steroidogenesis by CL from many different species (Armstrong, 1966; Marsh and Savard, 1966). Recent work has shown that CL formed following induced superovulation in prepuberal cattle contain progesterone, and that the plasma level of progesterone in these animals is considerably higher than that found in cows cycling normally (C. H. Spilman et al., unpublished). There are no reports, however, concerning the in vitro steroidogenic capabilities of CL from these animals. The present study was designed to measure the progesterone concentration of prepuberal calf CL obtained at various intervals after the induction of superovulation. The progesterone synthesizing capability of CL in' vitro, and the influence of luteinizing hormone (LH) and reduced nicotinamide-adenine din u c 1e o ti d e phosphate (NADPH) on this synthesis were measured. In addition, glucose utilization and lactate production by incubated CL were determined. Materials and Methods Fourteen Holstein calves, aged 3 to 5 months, were superovulated by a single intramuscular injection of 1,250 to 2,000 IU pregnant mares' serum gonadotropin (PMS) followed 5 days later by an intravenous injection of 75 rug LH. At 5, 10, 15 or 20 days after the ovulation inducing injection of LH, CL were collected at slaughter, sliced, and about 250 nag of tissue randomly distributed to each incubation flask. Usually two CL were selected at random from each animal. Each CL pro-

vided tissue for duplicate or triplicate incubations of all treatments. There were 2, 7, 8 and 8 CL, respectively, obtained on days 5, 10, 15 and 20. Tissue was incubated for 2 hr. at 37 to 38 C in either Krebs-Ringer bicarbonate buffer containing 1 mg/ml glucose (KRBG), K R B G + 5 /~g L H / m l (NIH-LH-B5), or KRBGq-0.7 x 10 3 M NADPH. Each incubation flask contained 4 ml medium and was gassed with 95% 0 2 - - 5 % CO2 before incubation. About 500 mg of each CL was immediately frozen and stored at --15 C for subsequent determination of initial progesterone concentration. After incubation the flasks were placed on dry ice to stop tissue metabolism and kept frozen until extraction procedures were begun. Upon thawing, 0.5 ml incubation medium was removed from each flask and glucose and lactate concentrations were determined enzymatically (Bergmeyer, 1965). The tissue and the remainder of the medium were transferred to a 200 ml round bottom flask, a tracer amount of progesterone-7-3H added, and the contents then extracted by refluxing in 95% ethanol according to Seifart and Hansel (1968). Progesterone was isolated by 2dimensional thin-layer chromatographic procedures (Armstrong, O'Brien and Greep, 1964), and quantified by UV absorption at 240 m~ using the Allen (1950) correction method. Appropriate corrections for sampling and for loss of tracer during purification were made. The data were statistically analyzed using analysis of variance. Standard errors shown in figures 1, 2 and 3 were caluculated from the error mean square of the analysis of variance. Results The average number of ovulations was

1 Department of Animal Science. 46 per animal. Gross observations of the CL .2Suggestions concerning the manuscript by Dr. W. Hansel and the technical assistance of Miss J. Wiebold and Mr. R. indicated that these glands were similar to Cole are appreciated. The hormones used were generously donated by Armour-Baldwin Laboratories (PLH), Ayerst those in mature heifers, but smaller than those Laboratories, Inc. (Equinex), and the Endocrinology Study following a single ovulation. The average Section, NIH ( N I H - L H - B 5 ) . This work was partially supported by NIH Grant HD 03471. weight per CL studied was 2.7 grams. At 5, 3Present address: Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545. 10 and 15 days after L H all CL in each ani+ Present address: Department of Physiology and Biophysics, mal were relatively uniform in appearance, Colorado State University, Ft. Collins 80521. 1025 JOURNAL OF ANIMAL SCIENCE, vol. 34, no. 6, 1972

SPILMAN E T AL.

1026 CALF CORPUS

LUTEUM

Effect

of LH

PROGESTERONE and

NADPH

SYNTHESIS

in v i t r o

200 [ " - 1 UNINCUBATED 180

J

CONTROL

LL~'~ t H o E

o) \ oi

&

tO e,J te.J t-

o to o o t-

J~

160

NADPH

140

120

I00

8O

///

o l=

///

60

~4

o o i.

I1.

4O

20

5 Days

after

hormonal

2O

15

10 induction

of

ovulation

F i g u r e 1. I n v i t r o p r o g e s t e r o n e s y n t h e s i s b y c o r p o r a l u t e a i n d u c e d i n p r e p u b e r a l calves. I n i t i a l p r o g e s t e r o n e concentration w a s m e a s u r e d in u n i n c u b a t e d tissue. S e p a r a t e C L t i s s u e w a s i n c u b a t e d in e i t h e r c o n t r o l K R B G , K R B G + L H or K R B G + N A D P H . O v e r a l l significant d i f f e r e n c e s : L H a n d N A D P H i n c u b a t i o n s w e r e g r e a t e r t h a n ( P ~ . 0 5 ) c o n t r o l i n c u b a t i o n s ; 5-, 10- a n d 15-day v a l u e s w e r e greater ( P ~ . 0 5 ) t h a n 20-day values. (T r e p r e s e n t s s t a n d a r d e r r o r . )

but after 20 days variation in size and gross appearance of CL, both within and among animals, indicated that asynchronous regression of luteal tissue was occurring. Initial luteal progesterone concentrations and the amount of progesterone after incubations are presented in figure 1. There was a small but significant ( P ~ . 0 5 ) decline in initial progesterone concentration with time. Asynchronous luteal regression that had been observed grossly was substantiated by the initial progesterone concentrations of 20-day CL (table 1). When two younger CL per calf were analyzed, progesterone concentrations (~g progesterone/g luteal tissue) were more alike, as indicated by the following paired comparisons: 40, 46; 10, 10; 36, 42; 49, 57; 18, 26; 33, 40; 24, 30; 28, 35. In all cases (figure 1), there was an increase in progester-

one concentration after control KRBG incubation. Tissue collected at 5, 10 and 15 days after L H synthesized similar amounts of progesterone, while 20-day CL produced only 41% as much progesterone. The CL in one 10-day animal had evidently regressed by this TABLE 1. I N I T I A L P R O G E S T E R O N E CONCENT R A T I O N OF D I F F E R E N T 20-DAY CORPORA L U T E A Progesterone (/zg/g)

Calf

CL1~

CL2

59 43 128 848

34 3 4 3

42 65 17 3

a CLx and CL were different corpora lutea taken from the same calf 20 days after the induction of superovulation. At this time asynchronous regression of CL was evident both within and among animals.

P R O G E S T E R O N E SYNTHESIS GLUCOSE METABOLISM

1027

BY C A L F C O R P O R A L U T E A IN V I T R O

7.0

6.0

~coNreot --I

o

5.0 ///

"x.

y//, 4.0 "o N

z//, 3.0 O U

2,0

1.0

///..~

5

10

15

20

Days afteP hormonal induction of ovulation

Figure 2. The effect of LH and NADPH on glucose utilization by incubated corpora lutea. NADlaH significantly increased (P(.01) glucose utilization by 5- and 15-day CL. (T represents standard error.) time; the initial luteal progesterone concentration was only 9.8/~g/g. The addition of L H to the incubation medium (figure l) stimulated progesterone synthesis only in 5-, 10- and 15-day CL. The magnitude of the response was much greater in 5-day CL. Evidently, 20-day CL were no longer capable of being stimulated by L H since the concentration of progesterone was identical to that after control K R B G incubation. Without exception, every incubation responded to N A D P H with increased progesterone synthesis. The response in 10-, 15- and 20-day CL incubations was greater than that induced by LH. As the CL aged its response to L H decreased while the effect of N A D P H remained essentially unchanged through day 15. Even at day 20, when incubated CL were no longer capable of responding to LH, they were stimulated to synthesize progesterone by NADPH.

Figures 2 and 3 present the results of glucose utilization and lactate production by incubated CL tissue. All incubations utilized glucose, but in no case was glycolytic activity stimulated by LH. However, N A D P H decreased the glucose in the medium when incubations contained CL collected at 5, 10 and 15 days. The magnitude of the response was much greater for 5- and 15-day CL than for 10-day CL. The increase following incubation of 10-day CL was due primarily to one incubation in which N A D P H more than doubled glucose utilization. Incubations containing 20-day CL were stimulated to synthesize progesterone by N A D P H , but were not stimulated to metabolize glucose. This age of CL and incubation treatment interaction was significant ( P ( . 0 5 ) . The accumulation of lactate in vitra was not affected by L H or by NADPH, even when N A D P H caused a 2-fold increase in glucose utilization (5- and 15-day tissue).

SPILMAN E T AL.

1028 LACTATE

PRODUCTION

BY C A L F C O R P O R A L U T E A

IN V I T R O

m CONTROL k~l LH J ~ NADPH

2.5-

2.0-

l

/

= E

._~

1

1.5-

1.0-

//,, /// /// /// 0.5-

/,I,

10 Days a f t e r

15

20

hormonal induction of ovulation

Figure 3. The effect of LH and NADPH on lactate production by incubated corpora lutea. LH and N A D P H had no effect (P~.05) on the accumulation of lactate in the incubation medium. 0" represents standard error.) Discussion

The superovulation treatment used here often resulted in more than 100 g of luteal tissue per animal. Despite the large amounts of luteal tissue, the concentrations of progesterone in unincubated luteal tissue were similar to those for CL from mature animals (Hafs and Armstrong, 1968). However, the progesterone levels in mature cattle followed a more cyclic pattern than was observed in the present study. All CL synthesized progesterone in control incubations, but the net synthesis was only about one-third of that obtained with CL from mature animals up to day 18 of the cycle (Armstrong and Black, 1966). After 20 days, slices of luteal tissue from prepuberal animals still synthesized progesterone in vitro without any exogenous gonadotropic stimulation. This is in contrast to luteal tissue slices from mature animals (Armstrong and Black, 1966), but in agreement with mature animal luteal tissue homogenates (Hafs and Armstrong, 1968). The net mass of progesterone synthesized when L H was added to the incubation medium

was somewhat less than that reported for mature cattle by Armstrong and Black (1966) and Armstrong, Lee and Miller (1970). Unlike data from mature animals (Armstrong and Black, 1966) the greatest effect of L H in the present study was on the youngest CL; 5-day luteal tissue synthesized more progesterone than did older CL. If L H acts preferentially on the most recently synthesized cholesterol (Armstrong and Black, 1968), then the greater response of 5-day luteal tissue may mean that this tissue contained more newly formed cholestro]. It is also possible that total cholesterol was limiting in older CL since de novo cholesterol synthesis may be inhibited by L H (Armstrong et al., 1970; Wilks, Fuller and Hansel, 1970). Progesterone production was stimulated by N A D P H in all cases. In agreement with the report by Armstrong and Black (1966) for luteal tissue from mature animals, 20-day CL incubations could still be stimulated by N A D P H regardless of the initial tissue concentration of progesterone and the fact that L H was without effect. Armstrong and Black (1968) suggested that the difference in effect

PROGESTERONE of L H and N A D P H on in vitro progesterone synthesis m a y be artifactual. N A D P H m a y not be able to reach the intracellular site of progesterone production, and its main effect m a y merely be on precursors which have leaked into the incubation medium from older, and possibly damaged, luteal cells. If so, the fact that calf CL responded to N A D P H in all incubations, m a y mean that these CL are more permeable to the co-factor or to proge.~terone precursors than are CL from mature ani reals. T h a t L H did not cause a significant increase in glycolysis in vitro is in contrast to the rep o r t of active CL from mature animals (Armstrong and Black, 1966). T w e n t y - d a y CL incubations were still stimulated to synthesize progesterone b y N A D P H , b u t were no longer stimulated to metabolize glucose. Therefore, an increase in glycolytic activity is not necessarily a prerequisite for, nor a consequence of, the stimulation in progesterone synthesis caused b y the addition of L H or N A D P H in vitro. Luteal regression begins in mature heifers between days 16 and 19 of the estrous cycle (Hansel and Snook, 1970), b u t the onset of luteal regression has not been studied previously in prepuberal cattle with an infantile reproductive tract. Regression clearly was occurring at 20 days, a time which is sooner than found in hysterectomized mature heifers b v Brunner, Donaldson and Hansel (1969). However, it is unknown whether or not the uterus of the calf contains luteolytic properties. The variable progesterone concentration in 20-day CL indicates that the initiation of luteal regression is asynchronous in these young animals. Since calves superovulated with P M S and human chorionic gonadotropin ovulate over a period of at least 48 hr. (Lineweaver and Hafez, 1970), asvnchronaus luteal regression m a y be the result of a heterogeneously aged population of CL. Summary Superovulation was induced in prepuberal calves using P M S and L H . The in~b;ced CL were collected at 5, 10, 15 or 20 days after the L H injection. Initial progesterone concentrations were greatest at 5 days, b u t remained high even to d a y 20. Upon incubation CL obtained after 5, 10 and 15 davs s'cnthesized more progesterone ( P < . 0 5 ) than 20-day CL in the control medium and in media containing L H or N A D P H . The overall

1029

SYNTHESIS

stimulation of progresterone synthesis b y L H was 27% and b y N A D P H was 60% when compared to control incubations. All incubated CL utilized glucose, b u t in no case was glycolysis stimulated b y L H . N A D P H stimulated glycolysis in 5-, 10- and 15-day CL incubations. I t did not stimulate glycolysis at 20 days even though these incubations could still be stimulated to synthesize progesterone b y N A D P H . The accumulation of lactate in vitro was not affected b y L H or b y N A D P H . Asynchronous regression of CL was observed both within and among animals a t 20 days, a fact which m a y in p a r t be due to different CL ages resulting from variable ovulation times in superovulated animals. Of major interest in these studies was the fact t h a t an abundance of functional luteal tissue was formed in prepuberal calves following superovulation. This tissue responded to L H and N A D P H in vitro b y increasing progesterone synthesis. Literature

Cited

Allen, W. M. 1950. A simple method for analyzing complicated absorption curves, of use in the colorimetric determination of urinary steroids. J. Clin. Endocrinol. 10:71. Armstrong, D. T. 1966. Comparative studies of the action of luteinizing hormone upon ovarian steroidogenesis. J. Reprod. Fertil. Suppl. 1:101. Armstrong, D. T. and D. L. Black. 1966. Influence of luteinizing hormone on corpus luteum metabolism and progesterone biosynthesis throughout the bovine estrous cycle. Endocrinol. 78:937. Armstrong, D. T. and D. L. Black. 1968. Control of progesterone biosynthesis in the bovine corpus luteum: effects of luteinizing hormone and of reduced nicotinamide-adenine dinucleotide phosphate in vitro. Can. I- Biochem. 46:1137. Armstrong, D. T., T. P. Lee and L. S. Miller. 1970. Stimulation of progesterone synthesis in bovine corpora lutea by luteinizing hormone in the presence of an inhibitor of cholesterol synthesis. Biol. Reprod. 2:29. Armstrong, D. T., J. O'Brien and R. O. Greep. 1964. Effects of luteinizing hormone on progestin biosynthesis in the luteinized rat ovary. Endocrinol. 75:488. Bergmeyer, H. V. 1965. Methods of Enzymatic Analysis. Academic Press, New York. Brunner, M. A., L. E. Donaldson and W. Hansel. 1969. Exogenous hormones and luteal function in hysterectomized and intact heifers. I. Dairy Sci. 52: 1849. Duncan, G. W., A. M. Bowerman, W. R. Hearn and R. M. Melampy. 1960. In vitro synthesis of progesterone by swine corpora lutea. Proc. Soc. Exp. Biol. Med. 104:17. Hafs, H. D. and D. T. Armstrong. 1968. Corpus luteum growth and progesterone synthesis during the bovine estrous cycle. J. Anita. Sci. 27:134. Hansel, W. and R. B. Snook. 1970. Pituitary ovarian relationships in the cow. J. Dairy Sci. 53:945.

1030

SPILMAN

Lineweaver, J. A. and E. S. E. Hafez. 1970. Ovarian responses in gonadotropin-treated calves. Amer. J. Vet. Res. 31:2157. Marsh, J. M. and K. Savard. 1966. Studies on the mode of action of luteinizing hormone on steroidogenesis in the corpus luteum in vitro. J. Reprod. Fertil. Suppl. 1 : 113.

E T AL. Seifart, K. H. and W. Hansel. 1968. Some characteristics and optimum incubation conditions of in vitro progesterone synthesis by bovine corpora lutea. Endocrinol. 82 : 232. Wilks, J. W., G. B. Fuller and W. Hansel. 1970. Role of cholesterol as a progestin percursor in rat, rabbit and bovine luteal tissue. Endocrinol. 87:581.