Decrease of various luteal enzyme activities during prostaglandin F2α-induced luteal regression in bovine

Moiecular and CeNular Endocrinoloig,

34 (1984) 99-105

99

Elsevier Scientific Publishers Ireland, Ltd. MCE 01097

Decrease of various luteal enzyme activities during prosta~andin F,,-induced luteal regression in bovine Ch.V. Rao ‘, J.J. Ireland 2 and J.F. Roche 3** ‘Departments of Obstetrics- Gynecology and Biochemistry, University of Louisville, School of Medicine,LouisviNe, KY 40292 (U.S.A.), ‘Department ofAnimal Sciences, Michigan State University, East Lansing, MI 48824 (U.S.A.), -‘Agricuiturat institute, Grange, County Meath ~Ireland~

(Received 17 August 1983; accepted 11 November 1983)

Keywords: estrous cycle; prostaglandin F,,; luteolysis, gonadotropin receptors; enzyme changes.

Summary Luteal gonadotropin receptors decrease in cows, sheep and rats within 24 h following an injection of a luteolytic dose of prostaglandin (PG) FZa. But it is not known whether this decrease is the specific event, or a reflection of general decline in luteal cell structure, function and metabolism. In order to investigate this possibi~ty, 15 of 21 heifers were given on day 9 of the estrous cycle, a single 500 pg injection of Cloprostenol (CO), a synthetic PGF,, analog. These heifers were ovariectomized in groups of 5 at 12, 24 and 36 h after CO. For controls, a group of 6 heifers were ovariectomized just prior to injection of the others. Serum progesterone levels decreased whereas LH levels increased (P < 0.05) by 12 h with no additional changes observed at 24 or 36 h. The luteal plasma membranes [‘251]hCG specific binding, as well as S-nucleotidase (5’-NE) activity, decreased by 12 h and continued to decline (P < 0.05) until 24 h (binding) or 36 h (5’-NE). Scatchard analysis showed that the decrease in ]‘2JIfhCG binding was due to a decrease in receptor number rather than a decrease in receptor affinity. The activities of cytochrome c oxidase in mitochondria, NADH cytochrome c reductase in rough endoplasmic reticulum and galactosyl transferase in Golgi decreased while NAD pyrophosphorylase in nuclei virtually disappeared following the injection of CO. The /3-N-acetyl-D-glucosaminidase (a lysosomal hydrolase) activity in the homogenate increased by 12 h and continued to increase up to 36 h. Glucose 6-phosphate dehydrogenase activity in cytosol was unchanged until 36 h after CO injection. We suggest from the above data that gonadotropin receptor decrease following CO injection is a reflection of general decline in luteal cell structural, functional and metabolic integrity.

Prostaglandin (PG) FZa of uterine origin was implicated in the initiation of luteolysis at the end of the bovine estrous cycle (Shemesh and Hansel, 1975; Peterson et al., 1975). A single injection of PGF,, can induce luteolysis at any time except * Present address: Veterinary Field Station, Ballycoolin Road, Finglas, Dublin 11 (Ireland) 0303-7207/84/!$03.00

during the first 4 days of the cycle (Rowson et al., 1972; Henricks et al., 1974). The structural, functional and biochemical events in exogenous PGF,,-induced luteolysis are similar to spontaneous luteolysis occurring at the end of the cycle (Umo, 1975; Stacy et al., 1976; McClellan et al., 1977; Spicer et al., 1981; Carlson et al., 1982). PGF,, treatment in vivo and in vitro antagonizes

0 1984 Elsevier Scientific Publishers Ireland, Ltd.

100

gonadotropin action in corpus luteum (Grinwich et al., 1976a; Thomas et al., 1978). This antagonism involves first a loss of gonadotropic responses, i.e. CAMP and progresterone production, followed by a loss of gonadotropin binding (Grinwich et al., 1976a, b; Thomas et al., 1978; Diekman et al., 1978). It therefore appears that, acute PGF,, action involves an uncoupling of regulatory (binding) and catalytic (adenyl cyclase) subunits of gonadotropin receptors, and chronic action involves a permanent loss of the gonadotropin binding component (Thomas et al., 1978). It is not known, however, whether the loss of gonadotropin binding is the specific event (in which case only the binding sites are lost) or a reflection of the generalized decline in luteal cell structure, function and metabolism. The present studies were undertaken to test the latter possibility by measuring various enzymes that reflect the structural, functional and metabolic integrity of luteal cells. The results show that most of the enzymes measured decrease in activity at the same time as gonadotropin binding. Materials and Methods Animals A detailed account of animal selection, the first Cloprostenol (500 pg) injection to synchronize estrous, the second Cloprostenol(500 pg) injection to study hormonal and biochemical events in induced luteolysis, the methods of ovariectomy and blood collection have already been described injection of (Spicer et al., 1981). A second Cloprostenol was given on day 9 of the estrous cycle to all heifers except 6 controls (0 h period). Groups of 5-6 heifers were ovariectomized at 0, 12, 24 and 36 h after the second injection of Cloprostenol. Iodination of hCG Unlabeled hCG (CR121, 13450 IU/mg, a gift from the National Pituitary Agency, NIAMDD) was radioiodinated by the lactoperoxidase technique (Miyachi et al., 1972; Rao et al., 1977). The specific activity of [ ‘251]hCG used in these studies varied from 79.1 to 88.5 pCi/pg. The maximal specific binding in the presence of excess plasma membranes ranged from 53.6% to 61.1% of the added hormone.

Subcellular fractionation Corpora lutea of control animals and of animals at various time points after Cloprostenol treatment were combined into three different pools, i.e. 3 pools for control, and 3 for each time point. The tissues were washed 3 times with homogenizing buffer (10 mM Tris-HCl, pH 7.0, containing 250 mM sucrose and 1 mM Ca’+), homogenized in the same buffer at 4°C with a Polytron homogenizer (PCU-2-110) at a setting of 6 using three 10 set bursts. The homogenates were filtered through 4 layers of cheesecloth, an aliquot was saved and the rest was subjected to centrifugation steps in order to obtain nuclei according to Chauveau et al. (1956) plasma membranes, mitochondria-lysosomes according to Gospodarowicz (1973) rough endoplasmic reticulum and Golgi fractions according to Ehrenreich et al. (1973) and cytosol. A flowchart (Fig. 1) is provided to supplement the above information and for the reader’s convenience. Following isolation, membranous organelles were washed once and resuspended in homogenizing buffer. All the fractions were stored in aliquots at -20°C. The protein content in aliquots of the fractions was determined following digestion according to Lowry et al. (1951) using bovine serum albumin as the standard. Binding studies Aliquots of 50 pg plasma membrane protein of all pools of corpora lutea were incubated for 2 h at 38°C with 0.1 nM [‘251]hCG in the presence and absence of lOOO-fold excess unlabeled hCG. After this initial evaluation of binding, plasma membranes of 0 and 12 h luteal pools were combined separately and incubated for 2 h at 38°C with increasing concentrations of [‘251]hCG (48 to 912 PM) in the presence and absence of 0.12 I_LM unlabeled hCG. The resulting specific binding data were transformed according to Scatchard (1949) and the binding constants were calculated. The other details on binding studies are the same as previously described (Rao et al., 1981). Enzyme assays The 5’-nucleotidase (EC 3.1.3.5) activity in plasma membranes was assayed according to Emmelot and Bos (1966) NAD pyrophosphorylase

101

HOMOGENATE kentr.

400X 8, 10 min I P

S

P (Discard)

RSP in HB, Centr.

S

Nuclei (Pellet)

Centr. 9000 X 8, 15 min

(Dizard)

I

I

S Centr.

Cytosol (Supernate)

105000 x 8, 2h

10 ml 9000 x 8 pellet in HB; 5 ml 308, 8 ml 36%, 8 ml 40% and 5 ml 50% sucrose, Centr. 66000x g, 1.5 h

I

P 10 ml 0.25 M, 8 ml 0.6 M, 10 ml 0.86 M and 10 ml of 1.15 M sucrose containing 105 000 X 8 pellet Centr. 63500X 8, 3.5 h

Plasma Membranes (Interface of 30-36s sucrose)

Rough Endoplasmic Reticulum (Pellet at bottom of tube)

Fig. 1. Flowchart for the subcellular supernate, P; pellet; HB, homogenizing

Mitochondria-Lysosomes (Interface of 4&50% sucrose)

and 36-40%

(Interfaces of 1.15-0.86 0.6-0.25 M sucrose)

M, 0.86-0.6

M;

fractionation of bovine corpora lutea. The abbreviations used are: buffer (see Materials and Methods for composition); RSP, resuspend.

(EC 2.7.7.1) in nuclei according to Kornberg (1950), cytochrome c oxidase (EC 1.9.3.1) in mitochondria-lysosomes according to Cooperstein and Lazarow (1951), NADH cytochrome c reductase (EC 1.6.99.3) in rough endoplasmic reticulum according to Mahler (1955) P-N-acetyl-D-glucosaminidase (EC 3.2.1.30) in homogenate according to Pugh et al. (1957), glucose 6-phosphate dehydrogenase (EC 1.1.1.49) in cytosol according to Kelly et al. (1955) and galactosyl transferase (EC 2.4.1.74) in Golgi according to Kim et al. (1971), with the exception that ovomucoid was used as an

Centr.,

centrifuge;

S,

acceptor protein. The inorganic phosphorus released in the 5’-nucleotidase assay was measured according to Fiske and Subbarow (1925) using KH,PO, as the standard. Briefly, the above enzyme activity measurements were based on: 5’-nucleotidase-the release of inorganic phosphate from 5’-adenosine monophosphate; NAD pyrophosphorylase-a two-step assay in which NAD formed during the first reaction from nicotinamide mononucleotide and ATP was quantified during the second reaction as NADH by an increase in absorbance at 340 nm;

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cytochrome c oxidasethe rate of oxidation of reduced cytochrome c as measured by an increase in absorbance at 550 nm; NADH cytochrome c reductase-the rate of reduction of oxidized cytochrome c in the presence of NADH; /?-N-acetylD-glucosaminidaserelease of phenol from phenyl-N-acetylglucosaminide as measured by an increase in absorbance at 650 hm; glucose 6-phosphate dehydrogenase-the rate of NADP reduction as measured by an increase in absorbance at 340 nm; galactosyl transferasetransfer of [ 3H]galactose from [ 3H]UDP galactose to monitored by liquid scintillation ovomucoid, counting. All enzyme activities were measured under optimal conditions with respect to time, temperature of incubation, concentration of various assay components and the amount of subcellular organelle protein. After subtraction of appropriate blanks, the enzyme-specific activities were calculated from linear rate data obtained on two different protein concentrations. Progesterone and LH measurements Serum levels of these two hormones were measured by their respective radioimmunoassays as previously described (Louis et al., 1973; Convey et al., 1976, 1977). Statistical analysis The values presented in this paper are the means and their standard errors of 1-4 observations multiplied by the number of animals or number of luteal tissue pools. One-way analysis of variance and either Fisher’s protected LSD mean test (Ott, 1977) or paired t test (Steel and Torrie, 1960) were used to test the significant changes.

LUTEAL WEIGHT IGRAMSI

PROGESTERONE

lng/rrdl

II

!

PLASMA

LH

(ng/mll

4 36

HOURS

AFTER

CLOPROSTENOL

Fig. 2. Luteal weights, peripheral luteinizing hormone levels following

INJECTION

serum progesterone and Cloprostenol injection.

and 36 h (5’-nucleotidase activity) following injection of Cloprostenol. Scatchard plot analysis (Fig. 4) of [‘*‘I]hCG after Cloprostenol injection revealed no differences in the receptor affinity (K, = 0.3 nM). However, the number of available receptors decreased (control = 19.0 fmoles/mg protein; treatment = 11.4 fmoles/mg protein) after treatment with Cloprostenol. The decrease in re1 ‘-

-hCG BINDING

5’.NUCLEOTIDASE

80

r’ 60

a F 5

40

b ae

Results 20.

Fig. 2 shows that, following Cloprostenol injection, luteal weights increased by 12 h and thereafter decreased (P < 0.05). Serum levels of progesterone decreased and LH increased (P -C0.05) by 12 h with no additional changes observed until 36 h. Fig. 3 shows that not only the [‘*‘I]hCG binding, but also the 5’-nucleotidase activity in plasma membranes, decreased (P < 0.05) by 12 h, with further decrease by 24 h ([‘251]hCG binding) or 24

PLASMA

LIL 2

24

HOURS AFTER

36

I

i

CLOPROSTENOL

I

!4

36

INJECTlON

Fig. 3. The [ ‘251]hCG specific binding and 5’-nucleotidase activity in luteal plasma membranes following Cloprostenol injection. The [ “*I]hCG bound (12.6 fmoIes/mg protein) and 5’-nucleotidase activity (309.0 nmoles of Pi released/min/mg protein) in controls were considered 100%.

103

ZIG

t-

18C

,-

; 15c P i? 8 I20 & z -90

60 2

I

3

psl- hCG WJND&M

30

Fig. 4. Scatchard analysis of [‘251]hCG specific binding to luteal plasma membranes of control animals (0) and of animals 12 h (EI) after Cloprostenol injection.

ceptors from 0 to 12 h may receptors by the increased receptors decreased rather 24 h in the absence of any This later decrease may be of receptors.

CIT-C

-0XtOASE

I i 12 24

be due to occupancy of LH levels. However, dramatically from 12 to further increase in LH. due to down regulation

NADH CfT- C REEUCTASE

NAD FfROPHOSPHORYLASE

ND I

36

12 24

36

NO”RS AFTER CLOPROSTENOL

12 24

36

-I_

INJECT&ON

Fig. 5. Cytochrome c oxidase, NADH cytochrome c reductase and NAD pyrophosphorylase activities following Cloprostenol injection. The activities in controls (cytochrome c oxidase, 38.3 pmoles of cytochrome c oxidized/min/mg protein; NADH cysochrome c reductase, 135.4 pmoles of cytochrome c reduced/min/mg protein; NAD pyrophospho~Iase. 2.9 pmoles of NAD formed/min/mg protein) were taken as 100%. ND, non-detectable. The values presented in this figure and in Fig. 6 represent the mean and range of duplicate measurements on the pooled fractions.

I2 24

36

12 24

36

Fig. 6. Galactosyl transferase, glucose 6-phosphate dehydrogenasa and ~-~-a~tyl-D-glucosa~nidase activities following Cioprostenol injection. The activities in controls (galactosyl transferase, 3.6 x lo6 dpm transferred/h/mg protein; glucose 6-phosphate dehydrogenase, 250.0 pmoles of NADPH formed/~n/mg protein; ~-~-a~tyl-D-~uc~a~nidase, 36.6 nmoles of phenol reIeased/min/mg protein) were considered 100%.

Fig. 5 shows the decrease in activities of mit~hondrial cyt~hrome c oxidase until 24 h and NADH cytochrome c reductase in rough endoplasmic reticulum until 36 h following Cloprostenol injection. Nuclear NAD pryophosphorylase, while detectable in controls, was undetectable at any time after Cloprostenol injection. Galactosyl transferase activity in Golgi was unchanged at 12 h but declined by 24 h (Fig. 6). The activity of cytosol glucose &phosphate dehydrogenase did not change until 36 h after Cloprostenol injection. The P-IV-acetyl-D-glucosaminidase (a lysosomal enzyme) activity in total homogenate progressively increased until 36 h after Cloprostenol injection. Discussion The animals for controls were ovariectomized just prior to CO injection of the others. Separate controls for each time point could not be run because of the limited availability of cows. Moreover, separate controls were not considered abso-

104

lutely essential since all the parameters measured in the present studies increase rather than decrease up to day 14 of the estrous cycle (Mitra et al., 1980). Serum progesterone levels and luteal gonadotropin receptors decreased by 12 h after Cloprostenol injection. Whether progesterone decreased first, followed by gonadotropin binding, or vice versa, is not known. However, it was not the intention of this study to obtain this information. Severai earlier studies have shown that progesterone decreases first, followed by a decrease in gonadotropin receptors (Grinwich et al., 1976a, b; Thomas et al., 1978; Diekman et al., 1978). The aim of this study was to investigate whether the decrease in luteal gonadotropin receptors following PGF;,, administration was a reflection of the general decline in luteal cell structural, functional and metabolic integrity. If this is true, then various enzymes that reflect these parameters should also decrease at the same time as the gonadotropin receptors. Enzymes used to evaluate this possibility reflect the status of every major organelle of the luteal cell. Needless to say, all these organelles contribute to different aspects of various biochemical events, including steroidogenesis. The enzymes of the steroidogenic pathway per se were not measured because the progesterone levels should reflect the status of at least some of the key enzymes involved in the progesterone biosynthetic pathway. The following putative roles can be ascribed to enzymes measured in this study. Adenosine, the end product of 5’-nucleotidase activity, is known to be a potent vasodilator (Burger and Lowenstein, 1970), and therefore may play a role in the regulation of luteal blood flow. Additionally, adenosine is shown to be a potent amplifier of gonadotropin action (Hall et al., 1981) and to antagonize PGF,, action (Behrman et al., 1982) in luteal tissue. Thus, its relative deficiency could lead to attenuation of gonadotropin action and enhancement of PGF,, action. Gafactosyl transferase plays a role in glycoprotein synthesis (Kim et al., 1971). The activities of glucose 6-phosphate dehydrogenase, cytochrome c oxidase and NADH cytochrome c reductase result in the production of cellular energy and reducing equivalents needed for steroid hydroxylations. The NAD, the end product of py-

rophosphorylase activity, serves as a cofactor for numerous cellular dehydrogenases and could play a role in regulating redox reactions in the cells (3uchwalow and Unger, 1977). The /3-N-acetyl-Dgiucosaminidase plays a role in degenerative changes during luteal regression. Most of the enzymes measured, as well as the gonadotropin receptors, decreased by 12 h after Cloprostenol injection. Some enzymes did not show a further decrease after 12 h whiie others continued to decrease until 24 or 36 h. Among the enzymes that decreased in activity, nuclear NAD pyrophosphorylase virtually disappeared by 12 h. Galactosyl transferase activity had not decreased at 12 h but did by 24 h. Glucose &phosphate dehydrogenase activity was unaltered up to 36 h. The N-acetyl-D-glucosaminidase activity in homogenate increased progressively up to 36 h after treatment. The present findings point out that: (a) The alterations in the enzyme activities were not indiscriminate. Some enzymes take a longer time to decrease, e.g. > 12 h for galactosyl transferase and possibly > 36 h for glucose 6-phosphate dehydrogenase. (b) NAD pyrophospho~Iase may perhaps be the most sensitive marker for luteolysis. (c) Lysosomal enzyme increase reflects an increase in the total number of lysosomes and/or an increase in the enzyme amount or activity of the existing lysosomes. The first possibility appears to be true, as previous ultrastructur~ studies reported an increase in the total number of lysosomes during spontaneous or PGF,,-induced luteolysis (Gemmell et al., 1976; McClellan et al., 1977). Whether gonadotropin receptors decreased first, followed by various enzymes, or vice versa, cannot be resolved from our data. However, it is unlikely that gonadotropin receptors decreased first because receptor decrease as measured in luteal total homogenates at 12 h did not reach statistical significance (Spicer et al., 1981), whereas the decrease measured in plasma membranes barely reached statistical significance (calculated t value = 2.5, tabular t value = 2.1 for 21 degrees of freedom). Previous studies have reported ultrastructural changes paralleling and consistent with declining

105

progesterone levels as early as 6 h after PGF,, administration (Stacy et al., 1976). This raises the possibility that enzyme activities may have decreased first. If the enzyme activities decreased first, or simultaneously with the gonadotropin receptors, then the suggestion that the receptor decrease is a reflection of structural, functional and metabolic changes of the luteal cell should be given serious consideration. Finally, some of the enzyme changes observed in this study are similar to those observed during spontaneous luteolysis (Mitra et al., 1980). Acknowledgements We gratefully acknowledge Dr. S. Mitra and Mr. Fred Carman, Jr., for performing various experiments. This work is supported by grants NIHHD-14697 (C.V.R.) and NSF PCM 7904571 (J.J.I.). References Behrman, H.R., Hall, A.K., Preston, S.L. and Gore, G.D. (1982) Endocrinology 110, 38-46. Buchwalow, I.B. and Unger, E. (1977) Exp. Cell Res. 106. 139-150. Burger, R.M. and Lowenstein, J.M. (1970) J. Biol. Chem. 245, 6274-6280. Carlson, J.C., Buhr, M.M., Wentworth, R. and Hansel, W. (1982) Endocrinology 110, 1472-1476. Chauveau, J., Mottle, Y. and RouiIIer, C.H. (1956) Exp. Cell Res. 11, 317-321. Convey, E.M., Beal, W.E., Seguin, B.E., Tannen, K.J. and Lin, Y.C. (1976) Proc. Sot. Exp. Biol. Med. 151, 84-88. Convey, E.M., Beck, T.W., Neitzel, R.R., Bostwick, E.F. and Hafs, H.D. (1977) J. Anim. Sci. 46, 792-796. Cooperstein, S.J. and Lazarow, A. (1951) J. Biol. Chem. 189, 665-670. Diekman, M.A., O’Callaghan, P., Nett, T.M. and Niswender, G.D. (1978) Biol. Reprod. 19, 1010-1013. Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 45-72. Emmelot, P. and Bos, C.J. (1966) Biochim. Biophys. Acta 120, 369-382. Fiske, C.H. and Subbarow, Y. (1925) J. Biol. Chem. 66, 375-400.

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