Isolated porcine ovarian follicles as a model for the study of
hormone and growth factor action on ovarian secretory activity
A V Sirotkin, A V Makarevich, J Kotwica1, P-G Marnet2,
H B Kwon3 and L Hetenyi
Research Institute of Animal Production, 949 92 Nitra, Slovak Republic, 1Institute of Animal Reproduction and Food Research, 10???718 Olsztyn-Kortowo,
Poland, 2Laboratoire de Recherches sur la Traite, INRA/ENSAR, 35042 Rennes Cedex, France and 3Department of Biology, Chonnam National University,
Kwangju 500???757, Korea
(Requests for offprints should be addressed to A V Sirotkin)
Abstract
The aim of our in vitro experiments with isolated
porcine ovarian follicles was to study the effects of
gonadotropins, GH, IGF-I and oxytocin (OT) on release
of ovarian steroid, OT, IGF-I, insulin-like growth factorbinding
protein-3 (IGFBP-3), prostaglandin F (PGF),
prostaglandin E (PGE) and cAMP.
It was found that quarters of ovarian follicles cultured for
8 days produced significant amounts of progesterone,
estradiol-17 beta, OT and IGFBP-3 with peaks of accumulation
from the 3rd to the 8th day of culture. Addition
of serum promoted progesterone, estradiol and OT release,
whilst accumulation of IGFBP-3 was maintained to a
greater extent in serum-free medium.
GH (10 ng/ml or above) was able to inhibit androstenedione,
OT, PGF and IGFBP-3, to stimulate IGF-I and
cAMP, and to alter testosterone and PGE release by
follicles cultured in serum-supplemented and/or serumfree
medium. IGF-I (10 ng/ml or more) inhibited androstenedione
and PGF secretion, stimulated testosterone,
estradiol, OT and cAMP production, but did not influence
progesterone, IGFBP-3 or PGE output in these conditions.
-------- >>>>>>OT (100 ng/ml) was able to inhibit androstenedione
and to stimulate testosterone, IGF-I, PGF and PGE,
but not estradiol or IGFBP-3 release. A stimulatory effect
of LH on progesterone and OT and an inhibitory
influence of LH on estradiol secretion in the serumsupplemented
medium were observed. FSH in these
conditions stimulated OT, but not progesterone or
estradiol secretion. <<<<<<<<<<<<<<----------------
The use of this experimental model suggests the involvement
of gonadotropins, OT, GH and IGF-I in the
control of ovarian steroid and nonapeptide hormone,
growth factor, growth factor-binding protein, prostaglandin
and cyclic nucleotide production. The stimulatory
effect of GH on IGF-I, and the stimulatory influence of
IGF-I on OT, as well as coincidence of the majority of
effects of IGF-I and OT, suggest the existence of a
GH???IGF-I???OT axis. On the other hand, the different
patterns of action of GH and IGF-I on OT, estrogen and
IGFBP-3 suggest that part of the GH effect on ovarian
cells is IGF-I independent.
Journal of Endocrinology (1998) 159, 313???321
Introduction
There is a growing body of evidence that ovarian function
is controlled not only by gonadotropins (Hillier 1991,
Sirotkin & Nitray 1994b, Sirotkin et al. 1994, Erickson
1995, Erickson & Danforth 1995), but also by other
substances of hypophysial or extra-hypophysial origin. In
particular, growth hormone (GH) ( Jia et al. 1986, Sirotkin
& Nitray 1994a, Sirotkin & Schaeffer 1995, Sirotkin
1996a), insulin-like growth factor-I (IGF-I) (Giudice
1992, Paton & Collins 1992, Spicer & Echternkamp 1995,
Sirotkin & Makarevich 1996), oxytocin (OT) (Wathes
1989, Schaeffer & Sirotkin 1995, Sirotkin 1995, 1996b,
Sirotkin et al. 1996) and prostaglandin (Dennfors et al.
1983, Satoh et al. 1984, Michael et al. 1994) can regulate
gonadotropin receptors, secretion of growth factors, steroid
and nonapeptide hormones, prostaglandin F2-alpha (PGF)
and cyclic nucleotides by rodent, bovine and human
ovarian cells.
In pigs, effects of gonadotropin (Hillier 1991, Sirotkin
& Nitray 1994b, Sirotkin et al. 1994, Erickson 1995,
Erickson & Danforth 1995), OT (Schaeffer & Sirotkin
1995, Sirotkin 1995, 1996b, Sirotkin et al. 1996) and
prostaglandin (Dodson & Watson 1979, Michael et al.
1994) on ovarian secretions have been reported. On the
other hand, there is very little information on the effects of
GH and IGF-I on ovarian secretion in this species. Only
an increase in IGF-I production after GH treatment, as
well as a stimulatory influence of both GH and IGF-I
on porcine ovarian steroidogenesis, has been reported
(Giudice 1992, Spicer & Echternkamp 1995). These data
suggest indirectly that IGF-I is a mediator of GH action on
313
Journal of Endocrinology (1998) 159, 313???321 ? 1998 Society for Endocrinology Printed in Great Britain
0022???0795/98/0159???313 $08.00/0
ovarian steroid secretion. On the other hand, the influence
of GH and IGF-I on porcine ovarian growth factorbinding
protein, OT, prostaglandin and cyclic nucleotides,
which may be also mediators of hormone and growth
factor action, has not yet been studied.
To understand the role of various peptide hormones and
related substances in the control of ovarian secretory
activity, as well as to obtain indirect evidence on their
possible inter-relationships, a comparison of their effects on
the secretion of various substances by porcine ovarian cells
is necessary.
Most in vitro studies of the ovary have been performed
on cultured granulosa cells. It is known, however, that the
complete, long-term production of ovarian secretions
requires functional integrity of the theca and granulosa
compartments of the follicle (Hillier 1991, Erickson 1995).
In an attempt to find an adequate model for the study of
ovarian steroidogenesis during follicular development and
after gonadotropin treatment, several procedures for the
culture of whole mouse (Boland et al. 1993, Spears et al.
1996), bovine (Staigmiller et al. 1982, Kruip & Dieleman
1989), sheep (Terlou et al. 1988, Mann et al. 1992) and
porcine (Dodson & Watson 1979) ovarian follicles have
been developed. In these systems, short-term (3 h to 2
days) perfusion with oxygenated serum-supplemented
medium is usual. These perfusion systems have been used
to study inter-relationships between gonadotropin, steroid
hormones and prostaglandins within the ovary. Follicles do
not appear to have been used previously for the study of
GH, growth factors and their binding proteins, OT or
intracellular messengers.
The aims of our study were: (1) to determine the value
of isolated quarters of porcine ovarian follicles for longterm
culture in serum-free or serum-supplemented
medium and the production of hormones and other
biologically active substances, and (2) to study the influence
of GH, IGF-I, OT, luteinizing hormone (LH) and
follicle-stimulating hormone (FSH) on growth factor,
growth factor-binding protein, steroid and nonapeptide
hormones, eicosanoid and cyclic nucleotide secretion by
these follicles.
Materials and Methods
Isolation and culture of ovarian follicles
Ovaries from non-cycling, Slovakian white gilts, 180 days
old, without visible reproductive abnormalities, were
obtained at a local slaughterhouse. Ovarian follicles
were collected and processed according to Golubev &
Zavertjajev (1989) with several modifications. Briefly, 1 h
after slaughter the tissues surrounding the ovary were
removed and the ovaries opened with scissors at the site of
entry of blood vessels. The connective tissues inside the
opened ovary were gradually broken with large surgical
forceps in order to access the follicles. Follicles 3 mm in
diameter without visible signs of atresia were pressed down
and separated from the surrounding connective tissues
with small forceps. They were collected in a Petri dish and
washed four times in sterile Dulbecco???s modified Eagle???s
medium/F-12 1:1 mixture (Sigma, St Louis, MO, USA)
supplemented with 10% heat-inactivated fetal calf serum
(FCS, University of Veterinary Medicine, Brno, Czech
Republic), and 1% antibiotic???antimycotic solution
(Sigma). The follicles were then cut with small scissors into
four equal parts, and washed gently, avoiding detachment
of granulosa cells from the follicular wall. Quarters of
follicles were placed individually in 24-well plates
(Beckton Dickinson GmbH, Heidelberg, Germany) with
2 ml medium. In the first series of experiments (studies of
hormone accumulation in follicle-conditioned medium)
follicles were cultured for 8 days with or without FCS at
37·5 )C in humidified air. The incubation medium was
collected on days 0, 1, 2, 3, 4, 6 and 8 of culture.
Microscopical examination of the cultures after 8 days
showed that granulosa cells remained attached to the
follicular wall; only insignificant numbers of cells started to
grow away separately. In the second series of experiments
(studies of hormone and growth factor effects on secretory
activity) follicles were cultured for 4 days in serumsupplemented
and, in some cases, also in serum-free
medium in the presence of recombinant porcine GH
(rpSTH, Research and Development, Pittman-Moore
Inc., Terre Haute, USA; 0, 10 or 0, 10, 100 or
1000 ng/ml), recombinant IGF-I (Calbiochem, Lucerne,
Switzerland; 0, 10 or 0, 1, 10 or 100 ng/ml), porcine
LH (National Hormone and Pituitary Program, Ogden
BioService Corp., Rockville, USA; 0 or 10 ng/ml),
porcine FSH (SPOFA, Prague, Czech Republic; 0 or
10 ng/ml) or synthetic OT (Sigma; 0 or 100 ng/ml). The
blank control was represented by medium cultured without
follicular tissue. After culture, the follicle quarters were
weighed and the medium stored at "20 )C until analysis.
Immunoassay
Hormone, growth factor-binding protein and prostaglandin
concentrations in 25???100 µl incubation medium were
determined in duplicate without extraction. Progesterone,
androstenedione, testosterone, estradiol and insulin-like
growth factor-binding protein-3 (IGFBP-3) were analyzed
using commercial IRMA kits from DSL (Webster,
TX, USA). The RIA kit for cAMP assay was from
Immunotech (Marseille, France). PGF and prostaglandin
E (PGE) were determined using an RIA kit from the
Institute of Isotopes (Budapest, Hungary) or the RIA
described previously (Cetta & Goetz 1982, Chang et al.
1995). IGF-I was measured according to the method of
Furlanetto et al. (1977). OT was determined using our
RIA or EIA described previously (Kotwica & Skarzynski
1993, Marnet et al. 1994). The characteristics of these
assays are presented in Table 1.
A V SIROTKIN and others · Hormone action on ovarian follicles 314
Journal of Endocrinology (1998) 159, 313???321