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Enzyme activities in early, mid and late luteal phase bovine corpora lutea
Vol. 97, No. 4, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
December 31, 1980
Pages 1555-1561
ENZYME ACTIVITIES IN EARLY, MID AND LATE LUTEAL PHASE BOVINE CORPORA LUTEA Satyabrata Mitra*, Ch. V. Rao*, and Michael J. Fields+ *Departments of 0bstetrics-Gynecology and Biochemistry University of Louisville, School of Medicine Louisville, Kentucky 40292 +Animal Science Department University of Florida Gainesville, Florida 32611
Received November i0, 1980 SUMMARY Several enzyme activities, which reflect structural and functional integrity, were measured in appropriate organelles isolated from bovine corpora lutea of early, mid and late luteal phase of the cycle. Glucose 6-phosphate dehydrogenase, lactate dehydrogenase, cytochrome c oxidase, NADH cytochrome c reductase, galactosyl transferase, 5'-nucleotidase (5'-NE) and NAD pyrophosphorylase activities were high during mid luteal phase when corpora lutea are mature with respect to structure and function. Whereas, N-acetylB-D-glucosaminidase activity was high during late luteal phase when corpora lutea were undergoing regression. Activities of all enzymes, except 5'-NE and recovery of several organelles from corpora lutea of late luteal phase were higher than those of early luteal phase. This suggests a conservation of structure and metabolic functions, at least until terminal stages of luteal regression. INTRODUCTION The ruptured ovarian Graffian follicle rapidly undergoes hypertrophy and hyperplasia to form the corpus luteum (1,2).
The newly formed corpus
luteum continues to develop in size and function until about midcycle and then regresses before the next ovulation (1,2).
This cyclical ovarian
function is observed only in the absence of pregnancy (1,2).
The above
sequence of cyclical ovarian events are common for all the mammalian species except for the days in the cycle when the corpus luteum is considered to be in early, mid and late luteal phases (1,2).
This exception is due to varia-
tions in cycle length and day of ovulation during the cycle (1,2).
The mean
length of the bovine estrous cycle is 21 days (estrus is day 0 and ovulation occurs within hours after estrus) and corpora lutea from days 3, 13, and 19
0006-291X/80/241555-07501.00/0 1555
Copyright © 1980 by Academtc Press, Inc. All rtghts o f reproduction m any form reserved.
VOI. 97, No. 4, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the cycle are considered to be in early, mid and late luteal phases (3). The assessment of biochemical events taking place in corpora lutea during these three luteal phases is not only very important but also a prerequisite for investigating the hormonal influence on these events.
The measurement of
various enzyme activities, which reflect structural and functional integrity of luteal ceils, should be an effective way to assess these biochemical events. However, there is a paucity of published data on this topic. MATERIALS AND METHODS Eight corpora lutea of day 3, 5 of day 13 and 6 of day 19 were collected from slaughtered cows whose estrus information was known. The corpora lutea were weighed and stored at -80 ° until analyzed. All corpora lutea of day 3 were pooled into two batches. These two batches and individual corpora lutea of days 13 and 19, were homogenized and subjected to centrifugation schemes to obtain nuclei, plasma membranes, mitochondria-lysosomes, rough endoplasmic reticulum, golgi and cytosol fractions (4, other references therein). Cytosol from day 3 corpora lutea was inadvertantly not saved. Protein content in an aliquot of the fractions was determined according to Lowry et ai (5) using bovine serum albumin as the standard. Peripheral blood samples, obtained frcm the cows at time of slaughter were used in determining plasma progesterone levels by radioimmunoassay (6). The 5'-nucleotidase (EC 3.1.3.5.) activity in plasma membranes was determined according to Emmelot and Bos (7); NAD pyrophosphorylase (EC 2.7. 7.1 ) activity in nuclei according to Kornberg (8); cytochrome c oxidase (EC 1.9.3.1 ) activity in mitochondria-lysosomes according to Cooperstein and Lazarow (9); NADH cytochrome c reductase (EC 1.6.99.3 ) activity in rough endoplasmic reticulum according to Mahier (i0) and galactosyl transferase (EC 2.4.1.74 ) activity in golgi according to Kim et al (ii) with the exception that ovomucoid was used as an acceptor protein. Lactate dehydrogenase (EC 1.1.1.27) (12), glucose 6-phosphate dehydrogenase (EC 1.1.1.49) (13) and N-acetyl-~-D-glucosaminidase (EC 3.2.1.30) (14) activities were measured in cytosol. The inorganic phosphate released in the 5'-nucleotidase assay was measured according to Fiske and Subbarow (15) using KH2PO4 as the standard. All enzyme activities were measured under optimal conditions with respect to time, temperature of incubation, concentration of various assay components and the amount of organelles protein. Each enzyme activity was measured at the same time on all organelles of different corpora lutea using the same amount of protein. Enzyme specific activities were calculated from linear rate data after subtraction of appropriate blank values. The data presented in this manuscript are the means and their standard errors of one (recovery data) to three (two observations for all enzymes and three for galactosyl transferase) observations on each organelle multiplied by the number of corpora lutea or batches used for fractionation. An exception to the above was golgi fraction which was pooled within the same stage of the cycle prior to galactosyl transferase assay. Analysis of variance and Duncan's multiple range test or Student's t test were used in determining the differences between groups (16). RESULTS Table I shows that there was no difference among early, mid and late
1556
Vol. 97, No. 4, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Recovery of various subcellular organelles from bovine corpora lutea of early, mid and late luteal phases
Day of cycle Organelle
13
3
19
% Recovery with respect to homogenate
Plasma membranes
1.7 + 0.i a
3.0 + 0.1 a
2.7 + 0.4a
Nuclei
4.5 + 1.0a
4.3 + 0.4a
2.5 + 0.3b
Mitochondria-lysosomes
3.2 + 0.5 a
2.9 ! 0.4 a
3.0 ! 0.6 a
Rough endoplasmic reticulum
4.6 + 0.4 a
1.8 + 0.2b
1.8 + 0.ib
Golgi
0.6 + 0.0a
1.5 ~ 0.i b
1.5 ~ 0.2b
43.1 ! 0.9 a
49.3 ! 8.6 a
Cytosol
-
a,b, values on the same line with different superscripts are different (P <0.05)
luteal phase corpora lutea with respect to the recoveries of plasma membranes and mitochondria-lysosomal
fraction.
Nuclei recovery from early and mid
luteal phase corpora lutea was significantly higher compared to late luteal phase.
The recovery of rough endoplasmic reticulum from early luteal phase
corpora lutea was significantly higher than from mid and late luteal phases. Golgi recovery from early luteal phase corpora lutea were significantly lower than those from mid and late luteal phase.
There was no differences between
mid and late luteal phase corpora lutea with respect to cytosol recovery. The 5'-nucleotidase, NAD p y ~ p h o s p h o ~ y l a s e, cytoohrome
d oxidase, NADH
cytochrome c reductase, galactosyl transferase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities in appropriate organelles from mid luteal phase corpora lutea were significantly higher than those in early or late luteal phases
(Table II).
All of these enzymes activities except
5'-nucleotidase in late luteal phase corpora lutea were also significantly higher than those in early luteal phase.
1557
N-acetyl-~-D-glucosaminidase acti-
Vol. 97, No. 4, 1 9 8 0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II Enzyme activities in bovine corpora lutea of early, mid and late luteal phases
Enzyme
Day of cycle 13
3
19
5'-nueleotidase (nmoles of Pi released /min/mg protein)
367.5 +
19.7 a
464.0 + 10.4 b
405.0 + I1.8 a
NAD pyrophosphorylase(pmoles formed/min/mg protein)
107.7 +
15.4 a
248.8 + 17.9 b
188.4 + 21.3 c
Cytoehrome c oxidase (pmoles oxidized/ min/mg protein)
52.1 +
5.2a
153.7 + 10.7 b
91.0 +
5.7 c
NADH cytochrome c reductase reduced/min/mg protein)
23.0 +
3.4 a
141.3 +
7.8b
62.3 +
4.5 c
0.4 +
0.1 a
1.6 +
0.0 b
1.0 +
0.0 c
Lactate dehydrogenase (pmoles of NAD formed/min/mg protein)
262.0 +
6.9 a
176.8 +
6.7b
Glucose-6-phosphate dehydrogenase (pmoles of NADPH formed/min/mg protein)
303.9 + 25.0 a
of NAD
(pmoles
Galactosyl transferase (107 dpm [3H]galactose transferred/hr/mg protein)
N-acetyl-8-D-glucosaminidase (nmoles of phenol released/min/mg protein)
94.1 +
4.3 a
a'b'C'values on the same line with different superscripts are different
210.7 + 22.9 b
185.1 +
(P <0.05)
vity in cytosol of late luteal phase corpora lutea was significantly higher than that found in mid luteal phase.
This enzyme activity in mitochondria-
lysosomal fraction of mid and late luteal phase corpora lutea, although less as compared to cytosol enzyme
(probably due to lysosomal breakdown),
the same pattern as enzyme in the cytosol
(data not shown).
showed
There was no
detectable activity of this enzyme in mitochondria-lysosomal
fraction of
early luteal phase corpora lutea.
DISCUSSION The luteal weights and peripheral plasma progesterone
levels in these
cows were consistent with the values expected for early, mid and late luteal phases.
Although progesterone
in late luteal phase declined to levels found
1558
9.1 b
Vol. 97, No. 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
in early luteal phase, the luteal weight remained significantly higher than in the early luteal phase. All
the enzymes measured except N-acetyl-B-D-glucosaminidase exhibited
high activity in corpora lutea at a time (mid luteal phase) when the corpora lutea are mature with respect to structure and function.
The latter enzyme
showed high activity in late luteal phase when the corpora lutea are actively regressing.
Although various enzyme activities parallel the increase in
luteal growth, function and decline, the magnitude of these changes do not exactly correspond to each other.
It is not known, however, whether such a
correspondence should be expected. The changes in enzyme activities in early, mid and late luteal phase corpora lutea did not parallel the recoveries of organelles with which these enzymes are associated.
This may indicate that organelle content in luteal
cells is regulated differently from that of the enzymes.
The activities of
various enzymes, except 5'-nucleotidase, and recoveries of some of the organelles from late luteal phase corpora lutea were higher than those from early luteal phase.
As pointed out before, the luteal weights in late luteal phase
were also higher than those of early luteal phase. together, may suggest a)
These two findings taken
enzyme activities (except 5'-nucleotidase) and
some of the organelle recoveries were correlated more with luteal weight than with progesterone and b) there was a conservation of structure and metabolic functions of luteal cells, at least until the terminal stages of regression. The corpora lutea of mid luteal phase attained a peak in terms of their weight and progesterone production.
The increased progesterone production by
luteal cells requires greater amounts of reducing equivalents for steroid hydroxylations and cellular energy.
The increased activities of glucose-6-
phosphate dehydrogenase, lactate dehydrogenase, cytochrome c oxidase and NADH cytochrome c reductase during mid luteal phase should be expected to contribute directly or indirectly to these requirements.
NAD pyrophosphory-
lase is a constituent of nuclear chromatin (17) and NAD, the product of this
1559
Vol. 97, No. 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
enzymatic reaction, serves as a cofactor for cell.
mumerous dehydrogenases in the
Thus this enzyme could play an important role in regulating redox
reactions of the cells (17). The product of 5'-nucleotidase activity, i.e., adenosine, is known to be a potent vasodialator and involved in the regulation of blood flow (18). Therefore, it is possible that the increased and decreased 5'-nucleotidase activity in mid and late luteal phase, respectively, contributed to the increased and decreased blood flow to corpora lutea which is known to occur during these time periods (19). There could also be other factors involved in blood flow regulation.
The high activity of galactosyl transferase, which
plays a role in glycoprotein synthesis (20), at a time when corpus luteum is at its peak in progesterone biosynthesis (mid luteal phase) may indicate that the increased glycoprotein synthesis is concomittant or coincidental with progesterone synthesis, at least from early to mid luteal phase.
The increa-
sed activity of N-acetyl-B-D-glucosaminidase which is one of the lysosomal hydrolases, in late luteal phase corpora lutea may reaffirm the concept that lysosomal enzymes play an essential role in luteal regression (21). ACKNOWLEDGEMENT The assistance of Dr. Ray Roberts in collection of the corpora lutea is gratefully acknowledged. REFERENCES i)
2)
3) 4) 5) 6) 7) 8) 9)
Everett, J.W. (1961) The mammalian female reproductive cycle and its controlling mechanisms. In : Sex and Internal Secretions, (Young, W.C., ed.) Vol. I, pp. 497-555, The Williams and Wilkins Co., Baltimore. Young, W.C. (1961) The mammalian ovary In : Sex and Internal Secretions. (Young, W.C., ed.) Vol. i, pp.449-496, The Williams and Wilkins Co., Baltimore. Rao, Ch. V., Estergreen, V.L., Carman, F.R., Jr. and Moss, G.E. (1979) Acta Endoer. 91, 529-537. Rao, Ch. V., Mitra, S., Sanfilippo, J., and Carman, F.R., Jr. (1981) Am. J. Obstet. Gynecol., In press. Lowry, O.JH., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Lopez-Barbella, S.R., Warnick, A.C., Wise, T.H., and Fields, M.J. (1979) J. Anim. Sci. 48, 1135-1142. Em~elot, P., and Bos, C.J. (1966) Biochim. Biophys. Acta 120, 369-382. Kornberg, A. (1950) J. Biol. Chem. 182, 779-793. Cooperstein, S.J., and Lazarow, A. (1951) J. Biol. Chem. 189, 665-670.
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Vol. 97, No. 4, 1980
i0) ii) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21)
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Mahler, H.R. (1955) Methods in Enzymology(Colowick, S.P., and Kaplan, N.O., eds.) Vol. II, pp. 688-693, Academic Press, New York. Kim, Y.S., Perdomo, J., and Nordberg, J. (1971) J. Biol. Chem. 246, 5466-5476. Worthington Enzyme Manual (1977) (Decker, L.A., ed.) pp. 19-20, Worthington Biochemical Corporation, Freehold, New Jersey. Kelly, T.J., Nielson, E.D., Johnson, R.B., and Vestling, C.S. (1955) J. Biol. Chem. 212, 545-554. Pugh, D., Leaback, D.H., and Walker, P.G. (1957) Biochem. J. 65, 464-469. Fiske, C.H.,and Subbarow, Y. (1925) J. Biol. Chem. 66, 375-400. Steel, R.G., and Torrie, J.H. (1960) Principles and Procedures of Statistics, McGraw-Hill Book Co., New York. 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. Niswender, G.D., Reimers, T.J., Diekman, M.A., and Nett, T.M. (1976) Biol. Reprod. 14, 64-81. Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P., and Palade, G.E. (1973) J. Cell Biol. 59, 45-72. McClellan, M.C., Abel, J.H., Jr., and Niswender, G.D. (1977) Biol. Reprod. 16, 499-512.
1561
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
December 31, 1980
Pages 1555-1561
ENZYME ACTIVITIES IN EARLY, MID AND LATE LUTEAL PHASE BOVINE CORPORA LUTEA Satyabrata Mitra*, Ch. V. Rao*, and Michael J. Fields+ *Departments of 0bstetrics-Gynecology and Biochemistry University of Louisville, School of Medicine Louisville, Kentucky 40292 +Animal Science Department University of Florida Gainesville, Florida 32611
Received November i0, 1980 SUMMARY Several enzyme activities, which reflect structural and functional integrity, were measured in appropriate organelles isolated from bovine corpora lutea of early, mid and late luteal phase of the cycle. Glucose 6-phosphate dehydrogenase, lactate dehydrogenase, cytochrome c oxidase, NADH cytochrome c reductase, galactosyl transferase, 5'-nucleotidase (5'-NE) and NAD pyrophosphorylase activities were high during mid luteal phase when corpora lutea are mature with respect to structure and function. Whereas, N-acetylB-D-glucosaminidase activity was high during late luteal phase when corpora lutea were undergoing regression. Activities of all enzymes, except 5'-NE and recovery of several organelles from corpora lutea of late luteal phase were higher than those of early luteal phase. This suggests a conservation of structure and metabolic functions, at least until terminal stages of luteal regression. INTRODUCTION The ruptured ovarian Graffian follicle rapidly undergoes hypertrophy and hyperplasia to form the corpus luteum (1,2).
The newly formed corpus
luteum continues to develop in size and function until about midcycle and then regresses before the next ovulation (1,2).
This cyclical ovarian
function is observed only in the absence of pregnancy (1,2).
The above
sequence of cyclical ovarian events are common for all the mammalian species except for the days in the cycle when the corpus luteum is considered to be in early, mid and late luteal phases (1,2).
This exception is due to varia-
tions in cycle length and day of ovulation during the cycle (1,2).
The mean
length of the bovine estrous cycle is 21 days (estrus is day 0 and ovulation occurs within hours after estrus) and corpora lutea from days 3, 13, and 19
0006-291X/80/241555-07501.00/0 1555
Copyright © 1980 by Academtc Press, Inc. All rtghts o f reproduction m any form reserved.
VOI. 97, No. 4, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the cycle are considered to be in early, mid and late luteal phases (3). The assessment of biochemical events taking place in corpora lutea during these three luteal phases is not only very important but also a prerequisite for investigating the hormonal influence on these events.
The measurement of
various enzyme activities, which reflect structural and functional integrity of luteal ceils, should be an effective way to assess these biochemical events. However, there is a paucity of published data on this topic. MATERIALS AND METHODS Eight corpora lutea of day 3, 5 of day 13 and 6 of day 19 were collected from slaughtered cows whose estrus information was known. The corpora lutea were weighed and stored at -80 ° until analyzed. All corpora lutea of day 3 were pooled into two batches. These two batches and individual corpora lutea of days 13 and 19, were homogenized and subjected to centrifugation schemes to obtain nuclei, plasma membranes, mitochondria-lysosomes, rough endoplasmic reticulum, golgi and cytosol fractions (4, other references therein). Cytosol from day 3 corpora lutea was inadvertantly not saved. Protein content in an aliquot of the fractions was determined according to Lowry et ai (5) using bovine serum albumin as the standard. Peripheral blood samples, obtained frcm the cows at time of slaughter were used in determining plasma progesterone levels by radioimmunoassay (6). The 5'-nucleotidase (EC 3.1.3.5.) activity in plasma membranes was determined according to Emmelot and Bos (7); NAD pyrophosphorylase (EC 2.7. 7.1 ) activity in nuclei according to Kornberg (8); cytochrome c oxidase (EC 1.9.3.1 ) activity in mitochondria-lysosomes according to Cooperstein and Lazarow (9); NADH cytochrome c reductase (EC 1.6.99.3 ) activity in rough endoplasmic reticulum according to Mahier (i0) and galactosyl transferase (EC 2.4.1.74 ) activity in golgi according to Kim et al (ii) with the exception that ovomucoid was used as an acceptor protein. Lactate dehydrogenase (EC 1.1.1.27) (12), glucose 6-phosphate dehydrogenase (EC 1.1.1.49) (13) and N-acetyl-~-D-glucosaminidase (EC 3.2.1.30) (14) activities were measured in cytosol. The inorganic phosphate released in the 5'-nucleotidase assay was measured according to Fiske and Subbarow (15) using KH2PO4 as the standard. All enzyme activities were measured under optimal conditions with respect to time, temperature of incubation, concentration of various assay components and the amount of organelles protein. Each enzyme activity was measured at the same time on all organelles of different corpora lutea using the same amount of protein. Enzyme specific activities were calculated from linear rate data after subtraction of appropriate blank values. The data presented in this manuscript are the means and their standard errors of one (recovery data) to three (two observations for all enzymes and three for galactosyl transferase) observations on each organelle multiplied by the number of corpora lutea or batches used for fractionation. An exception to the above was golgi fraction which was pooled within the same stage of the cycle prior to galactosyl transferase assay. Analysis of variance and Duncan's multiple range test or Student's t test were used in determining the differences between groups (16). RESULTS Table I shows that there was no difference among early, mid and late
1556
Vol. 97, No. 4, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Recovery of various subcellular organelles from bovine corpora lutea of early, mid and late luteal phases
Day of cycle Organelle
13
3
19
% Recovery with respect to homogenate
Plasma membranes
1.7 + 0.i a
3.0 + 0.1 a
2.7 + 0.4a
Nuclei
4.5 + 1.0a
4.3 + 0.4a
2.5 + 0.3b
Mitochondria-lysosomes
3.2 + 0.5 a
2.9 ! 0.4 a
3.0 ! 0.6 a
Rough endoplasmic reticulum
4.6 + 0.4 a
1.8 + 0.2b
1.8 + 0.ib
Golgi
0.6 + 0.0a
1.5 ~ 0.i b
1.5 ~ 0.2b
43.1 ! 0.9 a
49.3 ! 8.6 a
Cytosol
-
a,b, values on the same line with different superscripts are different (P <0.05)
luteal phase corpora lutea with respect to the recoveries of plasma membranes and mitochondria-lysosomal
fraction.
Nuclei recovery from early and mid
luteal phase corpora lutea was significantly higher compared to late luteal phase.
The recovery of rough endoplasmic reticulum from early luteal phase
corpora lutea was significantly higher than from mid and late luteal phases. Golgi recovery from early luteal phase corpora lutea were significantly lower than those from mid and late luteal phase.
There was no differences between
mid and late luteal phase corpora lutea with respect to cytosol recovery. The 5'-nucleotidase, NAD p y ~ p h o s p h o ~ y l a s e, cytoohrome
d oxidase, NADH
cytochrome c reductase, galactosyl transferase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities in appropriate organelles from mid luteal phase corpora lutea were significantly higher than those in early or late luteal phases
(Table II).
All of these enzymes activities except
5'-nucleotidase in late luteal phase corpora lutea were also significantly higher than those in early luteal phase.
1557
N-acetyl-~-D-glucosaminidase acti-
Vol. 97, No. 4, 1 9 8 0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II Enzyme activities in bovine corpora lutea of early, mid and late luteal phases
Enzyme
Day of cycle 13
3
19
5'-nueleotidase (nmoles of Pi released /min/mg protein)
367.5 +
19.7 a
464.0 + 10.4 b
405.0 + I1.8 a
NAD pyrophosphorylase(pmoles formed/min/mg protein)
107.7 +
15.4 a
248.8 + 17.9 b
188.4 + 21.3 c
Cytoehrome c oxidase (pmoles oxidized/ min/mg protein)
52.1 +
5.2a
153.7 + 10.7 b
91.0 +
5.7 c
NADH cytochrome c reductase reduced/min/mg protein)
23.0 +
3.4 a
141.3 +
7.8b
62.3 +
4.5 c
0.4 +
0.1 a
1.6 +
0.0 b
1.0 +
0.0 c
Lactate dehydrogenase (pmoles of NAD formed/min/mg protein)
262.0 +
6.9 a
176.8 +
6.7b
Glucose-6-phosphate dehydrogenase (pmoles of NADPH formed/min/mg protein)
303.9 + 25.0 a
of NAD
(pmoles
Galactosyl transferase (107 dpm [3H]galactose transferred/hr/mg protein)
N-acetyl-8-D-glucosaminidase (nmoles of phenol released/min/mg protein)
94.1 +
4.3 a
a'b'C'values on the same line with different superscripts are different
210.7 + 22.9 b
185.1 +
(P <0.05)
vity in cytosol of late luteal phase corpora lutea was significantly higher than that found in mid luteal phase.
This enzyme activity in mitochondria-
lysosomal fraction of mid and late luteal phase corpora lutea, although less as compared to cytosol enzyme
(probably due to lysosomal breakdown),
the same pattern as enzyme in the cytosol
(data not shown).
showed
There was no
detectable activity of this enzyme in mitochondria-lysosomal
fraction of
early luteal phase corpora lutea.
DISCUSSION The luteal weights and peripheral plasma progesterone
levels in these
cows were consistent with the values expected for early, mid and late luteal phases.
Although progesterone
in late luteal phase declined to levels found
1558
9.1 b
Vol. 97, No. 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
in early luteal phase, the luteal weight remained significantly higher than in the early luteal phase. All
the enzymes measured except N-acetyl-B-D-glucosaminidase exhibited
high activity in corpora lutea at a time (mid luteal phase) when the corpora lutea are mature with respect to structure and function.
The latter enzyme
showed high activity in late luteal phase when the corpora lutea are actively regressing.
Although various enzyme activities parallel the increase in
luteal growth, function and decline, the magnitude of these changes do not exactly correspond to each other.
It is not known, however, whether such a
correspondence should be expected. The changes in enzyme activities in early, mid and late luteal phase corpora lutea did not parallel the recoveries of organelles with which these enzymes are associated.
This may indicate that organelle content in luteal
cells is regulated differently from that of the enzymes.
The activities of
various enzymes, except 5'-nucleotidase, and recoveries of some of the organelles from late luteal phase corpora lutea were higher than those from early luteal phase.
As pointed out before, the luteal weights in late luteal phase
were also higher than those of early luteal phase. together, may suggest a)
These two findings taken
enzyme activities (except 5'-nucleotidase) and
some of the organelle recoveries were correlated more with luteal weight than with progesterone and b) there was a conservation of structure and metabolic functions of luteal cells, at least until the terminal stages of regression. The corpora lutea of mid luteal phase attained a peak in terms of their weight and progesterone production.
The increased progesterone production by
luteal cells requires greater amounts of reducing equivalents for steroid hydroxylations and cellular energy.
The increased activities of glucose-6-
phosphate dehydrogenase, lactate dehydrogenase, cytochrome c oxidase and NADH cytochrome c reductase during mid luteal phase should be expected to contribute directly or indirectly to these requirements.
NAD pyrophosphory-
lase is a constituent of nuclear chromatin (17) and NAD, the product of this
1559
Vol. 97, No. 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
enzymatic reaction, serves as a cofactor for cell.
mumerous dehydrogenases in the
Thus this enzyme could play an important role in regulating redox
reactions of the cells (17). The product of 5'-nucleotidase activity, i.e., adenosine, is known to be a potent vasodialator and involved in the regulation of blood flow (18). Therefore, it is possible that the increased and decreased 5'-nucleotidase activity in mid and late luteal phase, respectively, contributed to the increased and decreased blood flow to corpora lutea which is known to occur during these time periods (19). There could also be other factors involved in blood flow regulation.
The high activity of galactosyl transferase, which
plays a role in glycoprotein synthesis (20), at a time when corpus luteum is at its peak in progesterone biosynthesis (mid luteal phase) may indicate that the increased glycoprotein synthesis is concomittant or coincidental with progesterone synthesis, at least from early to mid luteal phase.
The increa-
sed activity of N-acetyl-B-D-glucosaminidase which is one of the lysosomal hydrolases, in late luteal phase corpora lutea may reaffirm the concept that lysosomal enzymes play an essential role in luteal regression (21). ACKNOWLEDGEMENT The assistance of Dr. Ray Roberts in collection of the corpora lutea is gratefully acknowledged. REFERENCES i)
2)
3) 4) 5) 6) 7) 8) 9)
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