289
Vol. 4, No. 3
ORIGINAL RESEARCH
Immunohistochemical localization
of androgen receptor and aromatase
in the ovary of the pregnant pig
Małgorzata Duda2, Małgorzata Burek2, Jerzy Galas2, Katarzyna Kozioł3,
Marek Koziorowski3, Maria Słomczyńska1,2
2
Laboratory of Endocrinology and Tissue Culture, Institute of Zoology,
Jagiellonian University, Kraków, 3Department of Physiology and
Reproduction of Animals, University of Rzeszów, Rzeszów, Poland
Received: 1 September 2004; accepted: 5 November 2004
SUMMARY
The distribution of androgen receptor (AR) and cytochrome P450 aromatase
was investigated in paraffin sections of pregnant pig ovary. Ovarian follicles
and corpora lutea were isolated from ovaries obtained on Days 10, 18, 32,
71 and 90 post coitum (p.c.). Androgen receptor was localized in the nuclei
of granulosa cells of follicles of various sizes. In addition, some follicles
demonstrated staining in the nuclei of theca interna cells. Stroma cells also
exhibited a positive immunostaining. At early and mid pregnancy (up to
Day 71) AR was expressed in the nuclei of luteal cells. Corpora lutea of Day
71 showed mainly cytoplasmic staining while on Day 90 almost all luteal
cells showed staining exclusively in the cytoplasm. Immunostaining for the
presence of cytochrome P450aromatase was very faint in all investigated
ovarian structures. The results could suggest that the process of androgen
1
Corresponding author: Laboratory of Endocrinology and Tissue Culture, Department of Animal
Physiology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Kraków, Poland;
e-mail: [email protected]
Copyright © 2004 by the Society for Biology of Reproduction
290
Ovarian AR and aromatase
aromatization plays a negligible role in the ovary of the pregnant pig. Reproductive Biology 2004 4(3): 289-298.
Key words: androgen receptor, aromatase, pig, pregnancy
INTRODUCTION
The androgen receptor (AR) together with estradiol, progesterone, and
mineralocorticoid receptors belongs to the large family of steroid hormone receptors which, after interaction with appropriate ligands serve
as transcription factors. By binding to hormone response elements and
forming functional complex with co-regulatory molecules they regulate
the transcriptional machinery [1]. Ligand-bound AR has been demonstrated to modulate uterine growth [15], stimulate prolactin secretion
[14], and antagonize the expression of estrogen-regulated genes [13].
Androgens are important modulators of the follicular function, and
interact with various factors to enhance granulosa cell differentiation.
On the other hand, androgens antagonize follicular development and
may induce apoptosis in granulosa cells. Androgens were reported to
enhance FSH-induced aromatase activity in cultured rat granulosa cells
and have a biphasic effect- with high concentrations being less potent
than lower ones.
Cytochrome P450 aromatase is a highly conserved enzyme encoded in
human beings by a single gene. Unlike other mammals, the pig expresses
functionally distinct izoenzymes encoded by three distinct genes [4, 6]. One
isozyme is expressed in the gonads, second in the porcine placenta while
the third is expressed in the early, pre-attachment porcine blastocyst [5].
The presence of AR and aromatase in the ovary has been demonstrated in
several species including humans, nonhuman primates, sheep and rats.
We have already demonstrated cyclic changes of AR and aromatase
immunostaining in the ovary of the cycling pig [16]. However, there is no
data on the location of these two proteins in the ovary of the pregnant pig.
Therefore, the aim of our work was to localize the androgen receptor and
cytochrome P450 aromatase in the follicles and corpora lutea of porcine
ovaries obtained on various days of pregnancy.
Duda et al.
291
MATERIALS AND METHODS
A polyclonal antibody against androgren receptors (NCL-Arp) was obtained
from Novocastra Lab, Newcastle-upon-Tyne, UK. Rabbit polyclonal antibodies against human placental cytochrome-P450 were a generous gift
from Dr. Yoshio Osawa (Hauptman-Woodward, Medical Research Institute,
Bufallo, NY). Biotynylated secondary antibodies were from Vector Lab,
Burlingame, CA, USA. Streptavidin-HRP complex was from Dako/AS,
Denmark. Paraplast was purchased from the Monoject Scientific Division
of Scherwood Medical, St. Louis, MO, USA. DPX medium was from Fluka.
All other chemicals were from Sigma-Aldrich.
Porcine ovaries were obtained from pregnant pigs on days 10, 18, 32,
71 and 90 post coitum (p.c.). Follicles and corpora lutea were fixed in 4%
paraformaldehyde, dehydrated in an increasing gradient of ethanol and
processed through paraplast. Immunohistochemistry was performed as
described in our earlier paper [16]. Finally, the sections were incubated
overnight with anti-AR (1:10) or anti-aromatase (1:300) antibodies followed
by biotynylated goat anti rabbit IgG (1:400) and streptavidin-horseradish
peroxidase complex (ABC/HRP; 1:100). The color reaction was developed
in TBS buffer pH 7.4 containing 0.01% H2O2, 0.05% diaminobenzidine,
0.07% imidazole. The primary antibodies were omitted or replaced by
normal rabbit serum in control sections. The sections were carefully rinsed
with Tris-buffered saline (TBS) pH 7.6 after each step of the described
procedure. Slides were dehydrated and mounted in DPX.
For each day of pregnancy, the material was obtained from two animals
and 10-20 follicles and 4-8 corpora lutea per ovary were used. To visualize
the morphology of follicles and corpora lutea, Goldner Trichrome staining
was performed on the selected slides. All procedures involving animals
were approved by the Animal Care and Use Committee.
RESULTS
Goldner Trichrome staining was used to visualize the morphology of follicles and corpora lutea obtained from swine ovaries at various days of
292
Ovarian AR and aromatase
Fig. 1. Follicles obtained from porcine ovaries of Day 10 post coitum (p.c.) A/
Morphology of structures visualized by trichrome Goldner staining; B/ Androgen
receptor was present in granulosa cells of the primordial as well as in the antral
follicles; C/ Very low level of immunostaining for cytochrome P450 aromatase
was observed in all components of the follicles (x 400; arrowhead- specific staining, asterisk – nonspecific staining).
Fig. 2. Follicles obtained from porcine ovaries of Day 18 p.c. A/ Morphology of
structures visualized by trichrome Goldner staining; B/ Very strong nuclear immunostaining for AR was observed in granulosa cells and stromal cells. Immunostaining was also observed in the theca interna layer; C/ Aromatase was absent from the
follicles (x 200; arrowhead- specific staining, asterisk – nonspecific staining).
Fig. 3. Follicles obtained from porcine ovaries of Day 90 p.c. A/ Trichrome Goldner
staining visualized the morphology of follicles; B/ Androgen receptor was localized
in granulosa, theca and stromal cells; the intensity of staining was very high; C/
Aromatase was still absent from the follicle (x 200; arrowhead- specific staining,
asterisk – nonspecific staining).
Duda et al.
293
Fig. 4. Corpus luteum (CL) dissected from porcine ovaries of Day 10 p.c. A/
Morphology of CL using trichrome Goldner staining; B/ Androgen receptor was
localized in the nuclei of the luteal cells; C/ No immunostaining of aromatase in
the luteal cells was observed (x 400; arrowhead- specific staining, asterisk – nonspecific staining).
Fig. 5. Corpus luteum (CL) isolated from porcine ovaries of Day 18 p.c. A/
Morphology of CL using trichrome Goldner staining; B/ Androgen receptor was
localized in the nuclei of the luteal cells; C/ No immunostaining of aromatase
in the luteal cells was observed. (x 400; arrowhead- specific staining, asterisk
– nonspecific staining).
Fig. 6. Corpus luteum (CL) isolated from porcine ovary of Day 90 p.c. A/ Morphology of CL using trichrome Goldner staining; B/ The pattern of AR staining
changed dramatically, AR was localized predominantly in the cytoplasm whereas
in follicular expression of AR remained nuclear; C/ Very weak immunostaining
of aromatase in some cells was observed (x 400; arrowhead- specific staining,
asterisk – nonspecific staining).
294
Ovarian AR and aromatase
pregnancy (fig. 1A-6 A). In the investigated ovarian structures, androgen
receptors were located predominantly in the nuclei of granulosa cells.
The intensity of AR immunostaining was dependent neither on the size
of the follicle nor the day of pregnancy (figs. 1B, 2B, 3B). Some thecal
cells also showed a positive staining. Positive reaction was observed in
stromal cells (fig.1B, 2B, 3B) in all investigated ovaries. Although in the
ovaries of pregnant pigs various types of follicles were present, the follicles exhibited similar pattern of AR immunostaining at the investigated
days of pregnancy.
Luteal cells were also positively stained but the staining was much
weaker (figs. 4B, 5B, 6B) than in granulosa cells. Similar to follicular cells,
AR was predominantly expressed in nuclei in luteal cells on days 10-32
p.c. In contrast, a shift of AR expression from the nuclei to the cytoplasm
was observed in the corpora lutea of Days 70 (not shown) and 90 p.c. It
is of interest that follicles from the same ovary still demonstrated strong
nuclear staining (fig. 6B).
The immunoreaction of aromatase in the ovary of pregnant pig was
neither present in the follicular cells nor in the luteal cells (fig.1C-5C). The
lack of aromatase protein was characteristic for follicles and corpora lutea
in all days of pregnancy except for luteal cells of Day 90 which showed a
very low level of aromatase. The omission of primary antibodies or incubation with normal rabbit serum resulted in a lack of immunostaining in
control slides.
DISCUSSION
Our earlier study on the ovary of cycling pigs [16] revealed that androgen
receptors are localized in the nuclei of granulosa cells and the intensity of
staining declined with the growth of follicles. In the follicles of pregnant
pigs, similar intensity of nuclear immunostaining was observed in different
days of pregnancy. In cycling animals, growth of ovarian follicles, ovulation
and formation of corpora lutea involve dramatic changes in their cellular
compartments. Steroid hormones which are produced by both follicles and
CL exert their role acting either through specific receptors or as substrates
Duda et al.
295
for steroidogenic enzymes. Changes in the distribution of steroid receptors
and aromatase were described in many species [8, 22] including pigs. In
the rat ovary [22] we observed that some proteins disappear (i.e. AR in the
preovulatory follicle) during the physiological reproductive cycle while
immunostaining for others increases (i.e. aromatase in the granulosa layer
of the preovulatory follicle).
Tetsuka et al [23] suggested that in order to create optimal conditions
for growing follicle, the action of androgens is at least partially regulated
at the receptor level. In the ovary of pregnant pig such phenomenon was
not observed. During pregnancy, follicles escape from the regulation by
gonadotropins and many of them undergo the process of atresia. Many
reports indicate that androgen can inhibit follicular development by increasing follicular atresia [3]. Atretic follicles demonstrated no decline in
AR and strong AR immunostaining was observed in their granulosa cells
[3]. However, one should keep in mind that some atretic follicles are luteinized and most likely consisted of hypertrophied theca interna cells and
already degenerated granulosa cells. Such follicles show a very faint AR
immunostaining. In contrast, the results of a study on AR-/- mice suggested
that AR may play an important role in granulosa cell survival during the
peri-ovulatory stage, and the absence of AR may cause granulosa cells to
be more susceptible to apoptosis [9].
The role of the androgen receptor in female fertility and ovarian function remains largely unknown. A recent study by Hu et al [9] demonstrated
longer estrus cycle and reduced fertility in AR-/- female mice in comparison
to AR+/+ animals. An observed marked reduction in the number of corpora
lutea suggests an insufficient granulosa cells number during follicle growth
in the AR-/- ovary. This may contribute to the impairment of CL formation.
Insufficient progesterone production caused a luteal phase defect. These
data prove that AR plays an important role in female reproduction. In mice
lacking AR, the authors observed reduced expression of FSH receptor gene
and insulin-like growth factor-I receptor which is essential for granulosa
luteinization [9].
The CL plays a crucial role in the maintenance of pregnancy in the pig
and androgens appear to be essential for the process of successful repro-
296
Ovarian AR and aromatase
duction. The in vitro study performed on corpora lutea from pregnant rats
showed that luteal cells from 15-day pregnant rats respond to androstenedione with increased progesterone production [2]. In vivo results confirmed
a direct luteotrophic effect of androstenedione in rat corpus luteum, not
mediated by previous conversion to estrogens. Experiments performed on
porcine luteal cells [7] showed that testosterone added to cells collected
in the mid-luteal phase of the cycle significantly increased progesterone
secretion by cultured cells.
The shift of AR protein from the nuclei to the cytoplasm which was
observed in luteal cells of late pregnancy (fig.6B) suggests a change in
androgen action. The ability to produce large amounts of estradiol is characteristic for the preovulatory follicles, which possess high amounts of
aromatase. Aromatase, which converts androgens to estrogens, is localized
in the cytoplasm. In the cycling pigs the immunostaining intensity reaches
the highest level in the preovulatory follicles [16] short before the LH surge.
However, all these associations change during pregnancy. Unlike in the
rat [21], ovaries of pregnant pigs do not produce estrogens. In the present
study, the immunohistochemical technique showed very clearly that almost
no aromatase protein was found in the ovary of pregnancy. Some positive
but weak staining was observed only in CL on Day 90. Increased level of
estrogens was observed in swine circulation after Day 70 of pregnancy but
it is known that, during this time the placenta remains the main source of
estrogens [10]. Between day 11 and 12 of gestation, pre-implantation pig
conceptuses produce estrogens, which are believed to be a critical component of the signaling mechanism for maternal recognition of pregnancy in
the pig [17, 18]. Androgens present in porcine uterine luminal fluid at early
pregnancy [19] may not solely serve as substrates for the production of E2
by pre-implantation conceptus, but may also modulate the biological effects of E2 in the endometrium [11]. The endocrine system of the sow must
gradually recover so that a new set of follicles can develop after weaning.
Under physiological conditions four to eight days after weaning, GnRH is
released from the hypothalamus and stimulates the pituitary to secrete LH
and FSH. Ovarian follicles begin to develop and secrete enough estradiol
to achieve estrus in the sow.
Duda et al.
297
The mechanism of the retrograde transfer of hormones has been analyzed
and described in detail [20]. The retrograde transfer in the peri-ovarian vascular complex of steroid hormones increases their concentration in blood
supplying the ovary [12, 19]. The increased level of estradiol, testosterone
and progesterone may modify, in a short loop of local feedback, the secretion of the ovarian steroids, both in the cycle and pregnancy. This local
mechanism should be taken into account in studies on the regulation of
ovarian function. In conclusion, androgen receptors present in the nuclei
of granulosa cells of the ovarian follicles may suggest that androgens are
essential for the maintenance of pregnancy.
ACKNOWLEDGMENTS
This study was supported by the State Committee for Scientific Research
as a Solicited Project PBZ-KBN-084/PO6/2002 from 2003 to 2005.
REFERENCES
1. Beato M Truss M Chavez S 1996 Control of transcription by steroid hormones. Annuals of New York Academy of Sciences 784 93-123.
2. Carizzo DG Rastrilla AM Telleria CM Aguado LI 1994 Androstenedione stimulates
progesterone production in corpora lutea of pregnant rats: an effect not mediated by
oestrogen. Journal of Steroid Biochemistry and Molecular Biology 51 191-197.
3. Cheng G Weihua Z Makinen S Makela S Saji S Warner M Gustafsson JA Hovatta O
2002 A role for the androgen receptor in follicular atresia of estrogen receptor beta
knockout mouse ovary. Biology of Reproduction 66 77-84.
4. Conley A Corbin J Smith T Hinshelwood M Liu Z Simpson E 1997 Porcine aromatase
studies on tissue-specific, functionally distinct isozymes from a single gene? Journal
of Steroid Biochemistry and Molecular Biolgy 61 407-413.
5. Corbin CJ Trant JM Walters KW Conley AJ 1999 Changes in testosterone metabolism
associated with the evolution of placental and gonadal isozymes of porcine aromatase
cytochrome P450. Endocrinology 140 5202-5210.
6. Graddy I Kowalski AA Simmen FA Davis SL Baumgarten WW Simmen RC 2000
Multiple isoforms of porcine aromatase are encoded by three distinct genes. Journal
of Steroid Biochemistry and Molecular Biology 73 49-57.
7. Gregoraszczuk E 1991 The secretion of testosterone and gonadotropins in stimulating
estradiol and progesterone secretion by cultures of corpus luteum cells isolated from
pigs in early and midluteal phase. Endocrinologia Japonica 38 229-237.
298
Ovarian AR and aromatase
8. Horie K Takakura K Fujiwara H Suginami H Liao S Mori T 1992 Immunohistochemical
localization of androgen receptor in the human ovary throughout the menstrual cycle in relation to estrogen and progesterone receptor expression. Human Reproduction 7 184-190.
9. Hu YC Wang PH Yeh S Wang RS Xie C Xu Q Zhou X Chao HT Tsai MY Chang C 2004
Subfertility and defective folliculogenesis in female mice lacking androgen receptor.
Proceedings of the National Academy of Sciences of the USA 101 11209-11214.
10. Knight JW Bazer F Thatcher WW Franke DE Wallace HD. 1977 Conceptus development in intact and unilaterally hysterectomized-ovariectomized gilts: Interrelations
between hormonal status, placenta development, fetal fluids and fetal growth. Journal
of Animal Science 44 620-637.
11. Kowalski AA Vale-Cruz DS Simmen FA Simmen RCM 2004 Uterine androgen receptors: Roles in estrogen-mediated gene expression and DNA synthesis. Biology of
Reproduction 70 1347-1357.
12. Krzymowski T Stefańczyk-Krzymowska S 2002 Uterine blood supply as a main factor involved in the regulation of the estrous cycle- a new theory. Reproductive Biology 2 93-114.
13. Lovely LP Rao KB Gui YT Lessey BA 2000 Characterization of androgen receptors
in a well-differentiated endometrial adenocarcinoma cell line (Ishikawa). Journal of
Steroid Biochemistry and Molecular Biology 74 235- 241.
14. Narukawa S Kanzaki H Inoue T Imai K Higuchi T Hatayama H Kariya M Mori T
1994 Androgens induce prolactin production by human endometrial stromal cells in
vitro. Journal of Clinical Endocrinology and Metabolism 78 165-168.
15. Sahlin L Norstedt H Eriksson H 1994 Androgen regulation of insulin-like growth
factor-I and the estrogen receptor in the rat uterus and liver. Journal of Steroid Biochemistry and Molecular Biolgy 51 57-66.
16. Słomczyńska M Tabarowski Z. 2001 Localization of androgen receptor and cytochrome
P450 aromatase in the follicle and corpus luteum of the porcine ovary. Animal Reproduction Science 65 127-134.
17. Spencer TE Bazer FW 2004 Conceptus signals for establishment and maintenance of
pregnancy. Reproductive Biology and Endocrinology 2 49-64.
18. Spencer TE Burghardt RC Johnson GA Bazer FW 2004 Conceptus signals for establishment and maintenance of pregnancy. Animal Reproduction Science 82-83 537-550.
19. Stefańczyk-Krzymowska S Grzegorzewski W Wąsowska B Skipor J Krzymowski T
1998 Local increase of ovarian steroid hormone concentration in blood supplying the
oviduct and uterus during early pregnancy of the sow. Theriogenology 50 1071-1080.
20. Stefańczyk-Krzymowska S, Wąsowska B, Chłopek J, Grzegorzewski W 2004 Retrograde transfer of steroid hormones to the ovary in luteal and follicular phases of
porcine oestrous cycle in vivo. Experimental Physiology 89 140-144.
21. Szołtys M Jabłonka A 1989 Hormonal function of rar ovarian tissues during pregnancy.
Progress in Zoology 35 103-105.
22. Szołtys M Słomczyńska M 2000 Changes in distribution of androgen receptor during maturation of rat ovarian follicles. Experimental and Clinical Endocrinology &
Diabetes 108 228-234.
23. Tetsuka M Whitelaw PF Bremner WJ Millar MR Smyth CD Hillier SG 1995 Developmental
regulation of androgen receptor in rat ovary. Journal of Endocrinology 145 535-543.