The present study was undertaken in order to characterize further the inhibitory activity of porcine follicular fluid. (FF1) upon the spontaneous maturation.
BIOLOGY
OF
REPRODUCTION
14,
Inhibition Fluid: ALEXANDER
511-516
(1976)
of Oocyte Partial
TSAFRIRI2,
Maturation
by Porcine
Characterization
SEYMOUR
Departments University
H.
POMERANTZ
of Physiology of Maryland 660 West Baltimore,
of the
Inhibitor’
and
and School
Redwood Maryland
Follicular
CORNELIA
P.
CHANNING3
Biochemistry, of Medicine,
Street, 21201
ABSTRACT The present study was undertaken in order to characterize further the inhibitory activity of porcine follicular fluid (FF1) upon the spontaneous maturation of isolated oocytes. The inhibitory action of FF1 upon oocyte maturation was not destroyed by heating to 60#{176}Cor by repeated freezing and thawing and could not be removed by extraction with charcoal. The inhibitory action was, however, abolished by trypsin digestion. The activity was concentrated by Amicon UM-2 membrane filtration. Further purification was achieved by the use of Sephadex G-25 column chromatography. The data are consistent with the suggestion that the inhibitor is a peptide with a molecular weight of about 2,000. The inhibitory activity of FF1 could be overcome by the addition of ovine LH (5 tg/ml) to the culture medium but not by bovine prolactin (10 Mg/mI) or dibutyryl cyclic AMP (0.04-0.4 mM). INTRODUCTION An upon
inhibitory the
oocytes
(1955).
We
of
action
of
porcine
porcine
to
effects
porcine cation
oocytes of
the
by
upon
the
the
characterize
FF1
upon to
the
achieve
isolated Chang inhibi-
and
of
has been demonstrated by us to support spontaneous maturation of porcine oocytes in culture (Tsafriri and Channing, 1975b). The FF1 was collected from 3-10 mm follicles and stored frozen until used for culture or fractionation. Similarly treated porcine serum (Grand Island Biological Co., Grand Island, N.Y.) was used for control incubations. The coilection and culture procedures utilized were similar to those
(FF1)
maturation
(Tsafriri
objectives
and
of
a similar
FF1
further of
fluid
described
oocytes The
were
tory
first
demonstrated
1975a).
study
follicular
maturation
was have
isolated
fling,
of
spontaneous
rabbit
tory
effect
the
Chartinhibi-
maturation a partial
previously described (Tsafriri and Channing, 1975a, b). The only change was the use of Falcon Microtest Ii plates instead of Lab-Tek slides for oocyte cultures. In each compartment of a Microtest Plate 10-15 oocytes were cultured in 0.2 ml of test medium. Hormones used in this study were ovine LH (NIH S19) and bovine prolactin (NIH B2). Dibutyryl cyclic AMP was purchased from the Boehringer Mannheim Co.
present
of purifi-
inhibitor.
MATERIALS
AND
METHODS Treatments
Oocyte
Collection
and
Culture
Oocytes isolated from porcine Graafian follicles (3-10 mm) were cultured, within their cumulus mass, for 45-48 h in medium 199 “Alex” containing the indicated proportion of untreated, fractionated, or treated FF1. Medium 199 “Alex” contains 15 percent pig serum, 12.5 mU/mi insulin, 0.03 mM pyruvate, and 2.5 mM lactate in a balance of medium 199 and
the Fort
‘Presented Society Collins,
in part at the 8th Annual Meeting for the Study of Reproduction held July 18-21, 1975, abstract 64.
of FF1
Aliquots of FF1 or serum were heated at 60#{176}Cfor 20 mm in a water bath or frozen and thawed repeatedly as indicated in Table 1. Charcoal extraction was performed by mixing 0.5 ml charcoal (Norite A; Fisher Chemical Co.) and 5 ml serum or FF1 in a graduated centrifuge tube for 10 mm, followed by centrifugation and sterilization of the supernatant by Millipore filtration. FF1 and serum were incubated for 1 h at 37#{176}Cwith 100 Mg/mI trypsin (2X crystallized. Sigma, St. Louis, Mo.). The trypsin treatment was terminated by the addition of soybean trypsin inhibitor (100 Mg/mI), Sigma, St. Louis, Mo). The treated FF1 or serum was used for the preparation of a 50 percent mixture with medium 199 “Alex” described previously (Tsafriri and Channing, 1975b).
of at
2Present address: Department of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel. 3Address reprint requests to: Dr. C. P. Channing, Department of Physiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, Maryland 21201.
Fractionation
of
FF1
Follicular fluid was separated into three fractions of different molecular size by filtering it consecutively through PM-10 and UM-2 Amicon membranes. The
511
TSAFRIRI
512
PM-b (NMW) a NMW
membrane has cut off of 10,000 cut off of 1,000.
Purification Inhibitor
of From
Oocyte
a
nominal the
and
molecular weight membrane has
UM-2
Maturation
FF1
A typical example of the purification was as follows: FF1 (340 ml; 20.1 g protein) was concentrated with nitrogen gas in an Amicon ultrafiltration cell using a PM-10 Amicon membrane until the retentate was a jelly-like mass. The PM-10 filtrate (246 ml; 46.7 mg protein) contained the bulk of the biological activity. The PM-b filtrate was then concentrated on a UM-2 Amicon membrane until the volume was 11.5 ml. At this point the concentration was interrupted and the UM-2 retentate was centrifuged at 105,000 X g for 30 mm to remove a precipitate. The clear supernatant was then further concentrated to a volume of 1.35 ml (3.51 mg protein; 0D280 = 6.3) and stored at -60#{176}C until used. 0.65 ml (1.75 mg protein) was loaded on a column of Sephadex G-25 (2 X 43 cm) suspended in a solution of 0.01 M sodium phosphate buffer, pH 6.8-0.10 M NaCI. This column had a void volume of 68 ml as judged with blue dextran. Porcine s3-melanocyte stimulating hormone (mol wt 2174) had a peak volume of 98 ml and 125j. appeared with a peak at 180 ml. The column was eluted with the suspension buffer and fractions of 2.5 ml were collected at the rate of 19-20 ml h1.
TABLE tion.
1. Effect
of
various
treatments
on
the
inhibitory
ET AL
Oocyte
Examination
and
Classification
At the end of the culture period the oocytes were collected and stained as previously described (Tsafriri and Channing, 1975a). The oocytes were divided into two groups: immature oocytes with an intact germinal vesicle and mature oocytes that had reached any stage beyond prometaphase (as described by Hunter and Polge, 1966), referred to as “total matured oocytes.” Within the mature group the percentage of oocytes in which the first polar body and the second metaphase chromosomes could be identified was indicated separately. The percentage of oocytes at any of the three described stages in each of the Microtest II plate compartments was calculated and the results were expressed as the mean ± SE of each experimental treatment. Degenerated oocytes never exceeded 10-15 percent of each group and were not indicated in the tables and in the figures. Differences between experimental groups were analyzed by the Student’s test.
RESULTS
FF1
Treatment Heating
freezing
of
FF1
and
tory
action
of
tion,
which
has
action
at
of porcine
Mean
60#{176}Cand
did (Table
thawing
FF1 been
up
not
to
6 cycles
affect
1).
foilicular
fluid
% of oocytes
inhibiextrac-
to
remove
Charcoal
demonstrated
upon
of
the
oocyte
matura-
(± SE) Mature
Treatment
A.
B.
C.
Freezing-thawing No. of times 1 3 6
frozen
Heating to 60#{176}Cfor Serum control FF1 control Serum heated FF1 heated
and
No. of oocytes
Dictyate
87 43 117
45.1 49.1 43.1
±
50 128 61 110
18.2 42.5 26.3 52.4
±
112 162 85 178
19.1 52.5 21.4 43.4
With polar
1st body
4.5 2.0 9.1
41.0 32.6 39.3
±
Total
thawed ± ±
6.0 2.4 9.1
51.7 42.1 53.1
±
l.8c 4.5 44b 4.3
75.0 53.0 63.6 42.6
±
4.8a 3.8 4.Oa 5.2
52.5 39.8 48.2 21.7
3.1 3.2 53a 5.0
71.0 44.3 74.4 52.1
± 2.7C
49.5 24.9 56.8 30.3
± ±
± ±
3.2 3.2 4.7
20 mm
Charcoal extraction Serum control FF1 control Serum extracted FF1 extracted
Porcine oocytes were cultured mination of the extent of oocyte aSerum vs corresponding FF1
for 48 maturation. P