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Biochemical and biological properties of urinary follicle-stimulating hormone (FSH) from the rhesus monkey (Macaca mulatta).

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American Journal of Primatology 27:205-213 (1992)
Biochemical and Biological Properties of Urinary
Follicle-Stimulating Hormone (FSH) From the Rhesus
Monkey (Macaca mulatta)
ROBERT L. MATTERI, PRADEEP K. WARIKOO, AND BARRY D. BAVISTER
Wisconsin Regional Primate Research Center, University of Wisconsin, Madison
Follicle-stimulating hormone (FSH) is routinely used for the induction of
superovulation in women. Homologous gonadotropin preparations that
could be used for reproductive studies in macaques would have valuable
research applications. Accordingly, we set out to characterize the physical
and biological characteristics of urinary FSH (UFSH) in the ovariectomized rhesus monkey. In urine from 7 monkeys, concentrations of bioactive FSH ranged from 16 to 57 Fgll, relative to cynFSH-RP1 (NIDDK).
UFSH was contrasted to pituitary FSH (PFSH) by non-reducing SDSpolyacrylamide gel electrophoresis (PAGE), native disc PAGE, and FPLC
chromatofocusing. The apparent molecular weights of UFSH and PFSH
are similar (approximately 35 kD); however, UFSH is more negatively
charged and demonstrates a lower overall isoelectric (pl) range than
PFSH. The bioactivity of UFSH was assessed by the stimulation of aromatase activity in cultured Sertoli cells and by induction of follicular
maturation in hamsters. Two fractions of pituitary FSH, which differed in
isoelectric properties, were obtained by chromatofocusing. The in vivo biological activity of FSH-A (acidic, pl3.8-4.6) and UFSH (pl3.5-4.5) were
similar, but greater than FSH-B (basic, pl4.6-5.5). These results support
the hypothesis that heavily sialylated, low pl FSH expresses high in vivo
bioactivity. This may reflect the well-known effect of sialic acid in prolonging the circulating half-life of glycoproteins. Thus, the quality and
quantity of FSH present in ovariectomized rhesus monkey urine indicates
that this may be a useful source for the preparation of enriched hormone
preparations. o 1992 Wiley-Liss, Inc.
Key words: superovulation, macaque, bioactivity
INTRODUCTION
There is a need for macaque follicle-stimulating hormone (FSH) that can be
used in studies in those species which require long-term or repetitive hormone
treatment. In macaques, the use of FSH from other species is known to induce the
formation of antibodies which neutralize the hormone [Platia et al., 1984;Bavister
h i v e d for publication April 8, 1991;revision accepted June 11, 1991.
Address reprint requests to Dr. Robert L. Matteri, Wisconsin Regional Primate Research Center, University of Wisconsin, 1223 Capitol Court, Madison, WI 53715-1299.
0 1992 Wiley-Liss, Inc.
206 I Matteri et al.
et al., 1986; Rotten et al., 1986; Wolf et al., 19901. Although 2 to 3 exogenous
stimulation procedures may be carried out in single monkeys, animals become
refractory after this point [Wolf et al., 19901. Therefore, there are serious concerns
in performing studies that require the repeated administration of heterologous
FSH for the induction of superovulation or the study of ovarian physiology in
macaques. Such studies would require a prohibitive number of animals to attain
statistically relevant results, produce results that could be influenced by immunological neutralization of hormone treatment, and have unknown effects on the
future reproductive status of valuable animals following experimentation.
In order to circumvent this problem, it is necessary t o identify and evaluate
potential sources of FSH. Based upon such information, hormone enrichment/
purification protocols can subsequently be developed. Since urinary gonadotropins
are routinely used for superovulation of women prior to artificial insemination or
in vitro fertilization, we were interested in evaluating urine from the rhesus monkey as a potential source of these hormones. As a first step in this process, we have
characterized the biochemical and biological properties of urinary FSH (UFSH) in
the ovariectomized rhesus monkey.
METHODS
Animals
Seven ovariectomized rhesus monkeys were housed at the Wisconsin Regional
Primate Research Center (WRPRC). The animals were kept a t 25-28°C under
controlled lighting conditions (14L:10D),and received standard pellet chow twice
daily with fruit supplement and tap water ad libitum. Overnight urine samples
were collected on ice from animals housed in metabolic cages.
Concentration of Urinary Proteins
Urinary protein was precipitated by the addition of solid ammonium sulfate
until a 75% saturated solution was achieved. This solution was kept at 4°C overnight. The precipitate was collected by centrifugation, redissolved in 0.05 M ammonium bicarbonate, desalted by Sephadex G-25 chromatography, and lyophilized.
A single monkey (rhesus 1662) was selected on the basis of having the highest
level of excreted bioactive FSH. One liter of urine was collected from this animal
and the protein harvested as above. This preparation is designated as UFSH. A
second concentration (UFSH-PoolC) was prepared from 1 liter of pooled urine from
5 animals.
Biochemical Analyses
Pituitary and urinary proteins were analyzed by FPLC chromatofocusing
[Matteri and Papkoff, 19881. A Mono-P column (Mono-P HR 5/20, Pharmacia) was
used in conjunction with an ISCO model 2350 HPLC pump (0.75 ml/min) and
model 2360 gradient programmer. One milligram of a pituitary protein extract
[Matteri and Ziegler, in press] was dissolved in 1 ml of 0.025 M ethanolamine, pH
9.5. The column was equilibrated with 0.025 M ethanolamine. The sample was
injected and 5 frictions were collected prior to beginning elution with 1:12 polybuffer 96 (Pharmacia), pH 5.5. After 20 min, the elution buffer was changed to a
1:lO solution of polybuffer 74 (Pharmacia), pH 3.5. Both polybuffer solutions contained 0.1% glycerol. When the pH gradient had stabilized, 1 ml 2 M sodium
acetate was injected to remove all material still adherent to the column (apparent
pl < 3.5). Fractions were collected into 12 X 75 glass tubes containing 0.75 ml
PBS-0.5% BSA, pH 7.5. The FSH content of each fraction was determined by RIA
(see below). Two fractions corresponding to the pl ranges of 4.6-5.5 (FSH-B) and
Urinary FSH in the Rhesus Monkey I 207
3.8-4.6 (FSH-A) were pooled, dialyzed, and lyophilized for subsequent biological
comparison to UFSH.
Electrophoretic evaluations of molecular weight, relative electric charge, and
isoelectric properties were performed in polyacrylamide gels, followed by immunological detection of FSH [Matteri & Ziegler, 19921.
Hormone Assays
In vitro FSH bioactivity was assessed as described previously [Matteri et al.,
19901. Reference preparation cynFSH-RP1 was obtained from the National Hormone and Pituitary Program (NHPP), NIDDK. Within- and between-assay variabilities were 5.6 and 8.1%, respectively.
The FSH radioimmunoassay (RIA) has been described previously [Hodgen et
al., 19761, with reagents provided by the NHPP. As with the bioassay, cynFSHRP1 was used as the reference preparation. Analysis of assay data was performed
by data reduction programs on the WRPRC VAX computer. Within- and betweenassay coefficients of variation were 4.2 and 9.6, respectively. Iodinations were
performed by the iodogen method [Matteri et al., 19871.
Stimulation of Follicular Development in Hamsters
In vivo FSH biopotency was determined in hamsters, using an endpoint of the
number of mature follicles produced [Bavister et al., 1986; Bavister, 19891. In
short, each female hamster weighing between 90 and 120 gm was injected intraperitoneally on the morning of post-estrous discharge (day 1) with a single
injection of FSH. Doses of FSH, as determined by the in vitro bioassay, ranged from
approximately 4 to 700 ng (see Fig. 5, Results). At least 2 hamsters were used at
each dose level. Fifty-three to 55 h after hormone injection (day 3), animals were
sacrificed by cervical dislocation. Their ovaries were removed and the number of
stimulated follicles was counted.
Statistics
In vivo bioactivity was plotted vs. in vitro potency for each FSH preparation.
Differences in the slopes of the regression lines, which represent in vivo vs. in vitro
biopotency, were detected by t-test [Glantz, 19811.
RESULTS
In the in vitro FSH bioassay, 1 vial (75 I.U.) of Pergonal was found to be as
potent as 20.3 rfr 3.1 pg cynFSH-RP1 (n = 3 assays). In order to determine the
quantity of FSH present in rhesus monkey urine, samples were obtained from
seven ovariectomized monkeys and analyzed for in vitro FSH bioactivity. Relative
to cynFSH-RP1 (NHPP, NIDDK), bioactive FSH concentrations ranged from 16 to
57 pg/l. In contrast, average bioactive FSH levels in urine from the follicular and
luteal phases of the rhesus monkey are less than 5 pg/l [Matteri et al., 19901.
Figure 1 demonstrates the FSH bioactivity present in the urine of three of the
animals.
The isoelectric properties of UFSH and PFSH were determined by chromatofocusing (Fig. 2). The UFSH demonstrated a more acidic isoelectric profile relative
to pituitary FSH. The hormone which remained bound to the column a t a pl of 3.5
was eluted with 2 M sodium acetate. This amounted to 3.4 and 8.7% of the total
hormone recovered from the chromatofocusing of PFSH and UFSH, respectively.
As predicted from their relative isoelectric characteristics, UFSH possessed
more material with high mobility than PFSH when analyzed by native disc gel
electrophoresis (Fig. 3). Similar data were obtained with UFSH-Pool C. As in the
208 I Matted et al.
10000
-
cynFSH-RPI
--%Y---
1662
I
u-91
1
AF-02
(u
W
m
n
100
:
.01
=
- -......1 - I
'
'.""I
1
-
-
'...'I
10
' ..-I
100
ng or ~IllWell
Fig. 1. Bioactive FSH concentrations in urine samples from 3 of the 7 ovariectomized monkeys utilized in the
present study. The reference preparation utilized was cynFSH-RP1 (NHPP, NIDDK estimated to be 15-20%
pure). Animal identification codes are indicated as U-91,AF-02,and 1662.
100
-
1
I
PFSH
UFSH
Y
0
Q)
P
3
4
5
7
6
8
9
10
PI
Fig. 2. Isoelectric resolution of immunoreactive urinary and pituitary FSH by FPLC chromatofocusing over a
pH gradient of 9.5to 3.5.Fractions between the pH values of 4.6 and 5.5 and 3.5 and 4.6were pooled to create
a basic and an acidic preparation of FSH (FSH-B and FSH-A, respectively).
chromatofocusing data, similar forms were present in both samples. However, the
relative proportions of these forms varied. Differences in isoelectric characteristics
between hormone preparations were not reflected in large differences in molecular
weight (Fig. 4).
The FSH present in urine appears to be fully bioactive in vitro, since the
hormone content of UFSH was determined as 0.248 and 0.246 pglmg by in vitro
-
Urinary FSH in the Rhesus Monkey / 209
100
80
V
UFSH
UFSH-PooIC
60
40
20
0
0.0
0.2
0.4
0.6
1 .o
0.8
Rf
Fig. 3. Native disc gel electrophoresis of pituitary and urinary FSH. While the overall charge properties vary,
similar forms appear in each preparation. Mobility is presented as migration relative to the dye front (Rf).
-
100
80
I_f_
FSH
UFSH-PooIC
60
40
20
0
0.0
0.2
0.4
0.6
0.8
1 .o
Rf
Fig. 4. Non-reducingSDS-PAGE [Matteri et al., 19901 of pituitary [PFSH] and urinary (UFSH, UFSH-Pool C)
FSH from the rhesus monkey. The FSH in all preparations was similar (approximately 35 M)). Mobility is
presented as migration relative to the dye front (FW.
bioassay and immunoassay, respectively (relative to cynFSH-RP1 reference). Considerable in vivo biopotency was also observed (Fig. 5). The relative in vivo vs. in
vitro activities of UFSH and UFSH-Pool C were similar to FSH-A, but greater
than FSH-B (P< 0.05;Fig. 5). The isoelectric properties of FSH-A and FSH-B were
evaluated by IEF-PAGE (Fig. 6 ) and found to correspond well to the chromatofocusing fractions from which they were derived.
210 I Matteri et al.
-
60
50
-
FSH-A
FSH-B
UFSH Pool'C'
40
TT/
30
20
1
10
100
1000
ng FSH
(In vltro potency rel. to cynFSH-RP1)
Fig. 5. The number of mature follicles resulting from the injection of various doses of urinary FSH (UFSH,
UFSH-Pool C), FSH-A (acidic PFSH), and FSH-B (basic PFSH) are plotted vs. in vitro biopotencies. The slopes
of the resulting regression lines were contrasted in order to assess relative in vivo and in vitro bioactivities.
DISCUSSION
Urinary FSH is produced commercially for the clinical induction of superovulation in women. Comparable gonadotropin preparations do not exist for
macaques. The ability to stimulate ovarian function repeatedly in these species
would permit the development of models for the study of human fertility and of
propagation of endangered nonhuman primates. Accordingly, the goal of the
present study was to evaluate the quantity, biochemical characteristics, and biological potency of urinary FSH in the rhesus monkey.
The ovariectomized rhesus monkey appears to excrete FSH in quantities
which could be utilized for ovarian stimulation. In the course of identifying animals with high levels of FSH excretion, we have found that up to 57 pg FSH/1 may
be present (relative to cynFSH-RP1). These levels significantly exceed those found
in the follicular and luteal phases of the rhesus monkey [Matteri et al., 19901. We
have determined 20 pg to be equivalent to one vial of 75 IU Pergonal (Serono Labs)
in the in vitro bioassay. It is known that the administration of 540 IU of Pergonal
over a 9 day period will induce follicular maturation in the rhesus monkey [Bavister, 19881. Depending on the animal used, this is the equivalent of approximately
3-6 1of urine with high FSH concentrations. While rhesus urinary FSH is highly
active in hamsters (Fig. 5), subsequent studies are needed to confirm ovarian
stimulation in macaques.
The present results indicate that UFSH possesses a high apparent negative
charge and low isoelectric profile. There is little doubt that this reflects an abun-
Urinary FSH in the Rhesus Monkey / 211
-
100
.
I
FSH-B
80
FSH-A
60
40
20
0
3
4
5
6
7
PI
Fig. 6. Isoelectric focusing (IEF) PAGE of FSH-A and FSH-B. Isoelectric characteristics of both preparations
correspond with the chromatofocusing fractions from which they were derived (Fig. 2).
dance of isoforms/isohormonesof FSH which contain a relatively large amount of
sialic acid [Chappel et al., 1983; Matteri & Papkoff, 19881. Isoforms of FSH with
high isoelectric points (pl) are known to be highly bioactive in terms of in vitro
biopotency and/or receptor binding activity [Chappel et al., 19831. While high pl
FSH isoforms are likely to possess a high degree of activity a t the target cell, these
forms are relatively deficient in sialic acid [Blum et al., 19851. Since sialic acid
prolongs the circulating half-life of glycoproteins [Morell et al., 1971; Ashwell &
Morell, 19741, it is not surprising that heavily sialylated, low pl forms of FSH are
metabolized relatively slowly [Blum & Gupta, 1985; Galway et al., 19901.This may
translate into a high degree of bioactivity in vivo which would not necessarily be
observed in vitro. Our present data, and those of Galway et al. [19901, support this
hypothesis (Fig. 4).
The biological quality of gonadotropins, as related to isohormone profile, is
influenced by a variety of physiological conditions. The qualitative properties of
secreted and/or intra-pituitary gonadotropins are influenced by GnRH [Dufau et
al., 1976; Resko et al., 19821, sex steroids [Peckham & Knobil, 1976a; Marut et al.,
1981; Chappel et al., 19841, gonadectomy [Peckham & Knobil, 1976a,bl, stage of
sexual development [Reader et al., 1983; Stroll0 et al., 1981; Wide & Hobson,
19831, and phase of the reproductive cycle [Adams et al., 1987; Cameron & Chappel, 1985; Robertson et al., 19791. It is expected, therefore, that the properties of
excreted gonadotropins also will be influenced by individual differences in endocrine status. The removal of steroid feedback produced by ovariectomy increases
the amount of gonadotropin secretion and the relative abundance of highly sialylated isohormones [Khan et al., 19851.In terms of quantity and quality, urine from
the ovariectomized rhesus monkey may offer a viable source of highly potent FSH.
Such gonadotropin preparations would have immediate research applications and
would serve as standards for the future production of recombinant FSH.
CONCLUSIONS
1. Ovariectomized rhesus monkeys secrete relatively high levels of bioactive
FSH.
212 I Matteri et al.
2. Urinary FSH from the ovariectomized monkey is highly charged and possesses a low pl, which is likely due to a relatively high sialic acid content.
3. The chemical and biological characteristics of urinary FSH produce further
evidence to support the concept that natural or recombinant FSH preparations to
be used for superovulation should be selected for a sialic acid content.
ACKNOWLEDGMENTS
We are grateful to Elaine Anderson for preparing this manuscript for publication. We wish to thank Frederick Wegner, Guenther Scheffler, and Daniel
Wittwer for their expert technical assistance. We also thank NIDDK and the
National Hormone and Pituitary Program (University of Maryland School of Medicine) for their gifts of hormones and reagents for assay purposes. This is Wisconsin
Regional Primate Research Center publication 31-002. This work was supported
by NIH grant RR00167 to the Wisconsin Regional Primate Research Center. This
study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals, NIH publication 85-23 and Public law 89-544, “The Animal Welfare Act,” August 24, 1966, and its amendments.
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