AABArchives Animal BreedingAABArch. Anim. Breed.2363-9822Copernicus PublicationsGöttingen, Germany10.5194/aab-60-131-2017The relationships between transforming growth factors β and free
thyroxine and progesterone in the ovarian cysts, preovulatory follicles,
and the serum of sowsStankiewiczTomasztomasz.stankiewicz@zut.edu.plWest Pomeranian University of Technology, Szczecin, Faculty of Biotechnology
and Animal Husbandry, Department of Animal Reproduction Biotechnology and
Environmental Hygiene, 29 Klemensa Janickiego Street, 71-270 Szczecin,
PolandTomasz Stankiewicz(tomasz.stankiewicz@zut.edu.pl)7June201760213113628October201629April20179May2017This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/3.0/This article is available from https://aab.copernicus.org/articles/60/131/2017/aab-60-131-2017.htmlThe full text article is available as a PDF file from https://aab.copernicus.org/articles/60/131/2017/aab-60-131-2017.pdf
The aim of the study was to determine the relationships between bone
morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9)
concentrations and free thyroxine (FT4) and progesterone (P4)
concentrations in follicular cysts, preovulatory follicles, and the serum of
sows (cyst-bearing (n=26) and non-cyst-bearing (n= 26)). FT4 and
P4 concentrations were higher in the cystic fluid than in the fluid of
preovulatory follicles (p< 0.01 and p< 0.05 respectively). BMP-15 and
GDF-9 concentrations were higher in the serum of cyst-bearing sows than
non-cyst-bearing sows (p< 0.05) and higher in the cystic fluid than in
the follicular fluid (p< 0.05). In the cysts and preovulatory follicles,
GDF-9 concentration was higher than in serum (p< 0.01). FT4
concentration in the serum of cystic sows was correlated with BMP-15
(r= 0.50, p< 0.05) and GDF-9 (r= 0.62, p< 0.01) concentrations in
serum. In the serum of non-cyst-bearing sows, a positive
correlation between P4 concentration and BMP-15 concentration
(r= 0.60, p< 0.01) was detected.
These data will help provide insight into the role of BMP-15, GDF-9,
FT4, and P4 during cyst formation in sows.
Introduction
Ovarian cysts account for a major proportion of ovarian dysfunction (Cech and
Dolezel, 2007; Szulańczyk-Mencel et al., 2010). In sows, polycystic
ovaries cause reproductive disorders, reduce reproductive performance, and
can result in their culling (Heinonen et al., 1998; Szulańczyk-Mencel et
al., 2010). Despite much research, the etiopathogenesis of ovarian cysts is
not yet fully understood. However, it is known that disturbances in hormonal
regulation can cause ovarian cysts (Kozłowska et al., 2013). Thyroid
hormones, as well as gonadotropins and steroid hormones, have important roles in
the regulation of the porcine ovarian follicle function (Maruo et al., 1987;
Gregoraszczuk et al., 1998) and are found in porcine ovarian follicular fluid
(Stankiewicz et al., 2008). In in vitro experiments, thyroid
hormones have been shown to affect steroidogenesis in porcine thecal and
granulosa cells (Gregoraszczuk and Skalka, 1996). The participation of
thyroid hormones in the synthesis of steroid hormones is noted by influencing
the activity of aromatase (Gregoraszczuk et al., 1998). It has been reported
that thyroid hormones increase the impact of the follicle-stimulating hormone on
the functional differentiation of cultured porcine granulosa cells (Maruo et
al., 1987). In addition, thyroid hormone receptors, their mRNA, or both have
been identified in porcine granulosa cells from preovulatory antral
follicles (Maruo et al., 1992). Thyroid status has also been implicated in
ovarian cyst formation in gilts (Fitko et al., 1995, 1996). Fitko et
al. (1995, 1996) have shown that hypothyroidism increases the exogenous
gonadotropin formation of cysts and weakens the steroidogenesis activity of
ovaries in gilts. However, exactly how thyroid hormones contribute to the
pathogenesis of ovarian cysts is unknown and might be based on interactions
in the ovaries and the central or peripheral interrelations. It is possible
that such interactions with thyroid hormones involve a bone morphogenetic
protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9), which belong
to the transforming growth factor β (TGF-β) superfamily. It is
supposed that BMP-15 and GDF-9 are involved in hormonal regulation of the
hypothalamic–pituitary–ovary axis (Paulini and Melo, 2011). These factors,
depending on the stage of follicular development, may increase or weaken the
influence of gonadotropins on the ovarian follicle (Knight and Glister, 2006;
Crawford and McNatty, 2012). In addition, they influence the proliferation
and differentiation of somatic cells of the follicle, steroidogenesis,
deposition of the extracellular matrix, ovulation, and luteinization (Su et
al., 2008; Orisaka et al., 2009; Peng et al., 2010). Also, recent work has
suggested a role for BMP-15 and GDF-9 in the pathogenesis of follicular cysts
in gilts and sows (Stankiewicz and Błaszczyk, 2014, 2016). However, the
exact activity of BMP-15 and GDF-9 in the pathogenesis of follicular cysts is
unknown.
Therefore, the objective of the research was to identify differences and
dependencies between BMP-15 and GDF-9 concentrations and the concentrations
of free thyroxine (FT4) and progesterone (P4) in follicular cysts,
preovulatory follicles, and the serum of sows (cyst-bearing and
non-cyst-bearing).
Material and methods
The study was carried out with Polish Large White × Polish Landrace
crossbreds (from 2 to 3 years old) slaughtered at a local slaughterhouse. All
sows were kept in a modern farm and then slaughtered in a modern, Polish
slaughterhouse according to national legislation and in line with European
Union legislation. At the time of slaughter, blood was collected from each
sow's cervical vein into a serum separator tube. The blood was centrifuged at
1000×g for 15 min, and the resulting serum was stored at
-20∘C until analysed. During an ongoing slaughtering process, 52
sows were chosen for further examinations, including 26 sows with follicular
cysts (cyst-bearing sows) and 26 sows without ovarian cysts but having
preovulatory follicles (non-cyst-bearing sows). Each sow was assigned one
fluid sample (from only one cyst or only one preovulatory follicle) and a
corresponding serum sample. In the experiment, the following groups
were distinguished for further research: follicular cysts (n= 26), preovulatory follicles
(n= 26), serum of cyst-bearing sows (n= 26), and serum of
non-cyst-bearing sows (n= 26). In order to obtain a uniform material, only
bilateral polycystic ovaries were examined in the experiment. In polycystic
ovaries, there were no corpora lutea and the current follicular structures
with thin walls were filled with fluid and had a diameter of more than 21 mm
(Heinonen et al., 1998; Cech and Dolezel, 2007; Sun et al., 2011). Ovaries in
the preovulatory phase were identified as those having at least several
follicles of the appropriate colour and composition, and they were 7 to 9 mm in
diameter (Hunter et al., 2004; Paradis et al., 2009). In these ovaries a single
corpus haemorrhagicum or corpus luteum was present. Ovaries with
corpora albicantia were eliminated from the study (Babalola and Shapiro,
1988). The follicular/cystic fluid was aspirated with a needle and syringe
from the preovulatory follicle/cyst. Fluid samples were centrifuged at 3000×g for 10 min to remove cellular material, and the supernatant was
stored at -20∘C until being analysed.
BMP-15 and GDF-9 assay
Specimen-specific kits were used to determine the concentration of BMP-15
(Porcine BMP-15 ELISA kit, Novateinbio Biosciences, Cat. No. POR10362) and
GDF-9 (Porcine GDF-9 ELISA kit, Novateinbio Biosciences, Cat. No.
BG-POR11087). The test sensitivities were 0.1 ng mL-1 and
0.1 pg mL-1 for BMP-15 and GDF-9 respectively. The intra- and
inter-assay coefficients of variation were < 10 % for BMP-15 and
GDF-9. The measurement was conducted using a Wallac fluorometer 1420
VICTOR2 (Wallac Oy, Turku, Finland). All assays were carried out in
duplicate.
FT4, P4, BMP-15, and GDF-9 concentrations in the ovarian
cystic fluid, preovulatory follicular fluid, and the serum of sows.
Correlation coefficients (r) between FT4 and P4
concentrations and the concentration of the transforming growth factors (BMP-15
and GDF-9) in the cystic fluid and serum of cyst-bearing sows (n=26).
ParameterFT4 in serumFT4 in cystic fluidP4 in serumP4 in cystic fluidBMP-15 in serum0.50*0.23-0.090.12BMP-15 in cystic fluid0.020.06-0.180.14GDF-9 in serum0.62**0.130.120.20GDF-9 in cystic fluid0.110.18-0.170.11
Values marked * and ** are significant at p<0.05 and
p<0.01 respectively.
FT4 and P4 assay
FT4 concentrations were measured by fluoroimmunoassay using the
Delfia® FT4 kit (Perkin-Elmer, Wallac
Oy, Finland) (Błaszczyk et al., 2006). The intra- and inter-assay
coefficients of variation were 3.3 and 4.7 % respectively. The
sensitivity of the assay was 1.56 pg mL-1. The P4 concentration
was also determined by fluoroimmunoassay using the
Delfia® P4 kit (Perkin-Elmer, Wallac Oy,
Finland) (Stankiewicz et al., 2008, 2009; Błaszczyk et al., 2009). The
intra- and inter-assay coefficients of variation were 4.9 and 6.9 %
respectively. The sensitivity of the assay was 0.25 ng mL-1.
All assays were carried out in duplicate. The measurements were made using a
Wallac fluorometer 1420 VICTOR2 (Wallac Oy, Turku, Finland).
Statistical analysis
The data are presented as an average ± standard deviation of the mean
and presented in the tables. Analysis of variance (ANOVA) and a post hoc test
was done to identify statistically significant differences. Duncan's multiple
range test was used to verify the significance of differences at p< 0.01
and p< 0.05. In addition, correlations between the analysed parameters
were calculated with the Spearmen's rank correlation coefficient. Statistical
analyses were conducted using the STATISTICA version 7.1, Stat Soft, Poland.
Results
Table 1 shows the mean concentrations of FT4, P4, BMP-15, and GDF-9
in cyst-bearing and non-cyst-bearing sows. The FT4 concentration in the
serum of cyst-bearing sows was not statistically different from its in-cyst
concentration. Also, FT4 concentration did not differ between the serum
and preovulatory follicles of non-cyst-bearing sows nor were any differences found between the FT4 concentration in the serum of cyst-bearing
sows and non-cyst-bearing sows. However, the FT4 concentration in the
cystic fluid was significantly higher than in the fluid of preovulatory
follicles.
In cyst-bearing and non-cyst-bearing sows, the P4 concentration was
significantly higher in the ovary structures (cysts and preovulatory
follicles) than in the serum. Also, P4 concentration was significantly
higher in cysts than in preovulatory follicles.
BMP-15 and GDF-9 concentrations were significantly higher in the serum of
cyst-bearing than in that of non-cyst-bearing sows. BMP-15 and GDF-9 concentrations were
also higher in the cystic fluid than in follicular fluid. BMP-15
concentration in the preovulatory follicles did not differ significantly
from its concentration in serum. However, these values did differ for GDF-9.
In the preovulatory follicles, GDF-9 concentration was significantly higher
than in the serum. Similar differences were found in cyst-bearing sows.
BMP-15 concentration in cysts did not differ significantly from its
concentration in serum. However, GDF-9 concentration in cysts was
significantly higher than in serum.
Correlation coefficients (r) between FT4 and P4 concentrations and
the concentration of the transforming growth factors (BMP-15 and GDF-9) in
the preovulatory follicle fluid and serum of non-cysts-bearing sows (n=26).
ParameterFT4 in serumFT4 in follicle fluidP4 in serumP4 in follicle fluidBMP-15 in serum0.280.200.60*0.19BMP-15 in follicle fluid0.390.350.24-0.18GDF-9 in serum0.02-0.05-0.01-0.24GDF-9 in follicle fluid-0.14-0.110.310.40
Values marked * are significant at p<0.01.
Tables 2 and 3 show the correlation coefficients between FT4, P4, and transforming growth factors β in cyst-bearing and non-cyst-bearing
sows. In the serum of cystic sows, the FT4 concentration was positively
and significantly correlated with BMP-15 and GDF-9 concentrations. In
non-cyst-bearing sows, a positive and significant correlation was found
between serum P4 and BMP-15 concentrations.
Discussion
Studies on the pathogenesis of ovarian cysts must consider many potential
contributing factors (Fitko et al., 1995, 1996; Kozłowska et al., 2013;
Pierre et al., 2016). As such, it is not yet possible to exclude
contributions by BMP-15 and GDF-9 during cyst formation (Stankiewicz and
Błaszczyk, 2014, 2016). Increased BMP-15 and GDF-9 concentrations were
found in the follicular cysts of gilts and sows (Stankiewicz and Błaszczyk,
2014, 2016). Here, I detected higher BMP-15 and GDF-9 concentrations in the
cystic fluid than in follicular fluid. However, these differences were
smaller than in earlier work (Stankiewicz and Błaszczyk, 2014, 2016).
Nevertheless, the presence of BMP-15 and GDF-9 in the follicular and cystic
fluid confirms that these factors create the proper follicular
microenvironment and can participate in the development of follicular cysts.
In addition, BMP-15 and GDF-9 concentrations are positively correlated in
follicular fluid and serum (Stankiewicz and Błaszczyk, 2016). Thus, I
cannot exclude their participation in the peripheral control of
folliculogenesis.
As shown here, the concentration of BMP-15 and GDF-9 in serum is higher in
cyst-bearing than non-cyst-bearing sows, which is in line with previous studies
(Stankiewicz and Błaszczyk, 2016). Unlike in previous studies, in the
current study it was also found that GDF-9 concentration was higher in the
cystic and follicular fluid than in serum. The higher concentrations in the
cystic and follicular fluids show, that the synthesis of transforming growth factors
such as GDF-9 and BMP-15 occurs in ovarian follicles at various stages of
development. According to Fitzpatrick et al. (1998) and Knight and
Glister (2006), the expression of GDF-9 mRNA was found in the hypothalamus,
pituitary, and uterus of different mammalian species, so that theses
extraovarian organs can also influence the serum levels. The exact role of
BMP-15 and GDF-9 in the pathogenesis of follicular cysts is unknown.
Therefore, in the present study, I aimed to define the relationships between
these and other factors that might participate in the formation of follicular
cysts. One of such factor is the steroidogenic activity of the ovarian
follicle. Disturbed follicular steroidogenesis is either an effect or a cause of ovarian disorders, such as ovarian cysts (Babalola and Shapiro,
1990; Szulańczyk-Mencel et al., 2010). Here, higher levels of P4
were found in the cystic fluid than in preovulatory follicle fluid, which is
in line with previous studies (Babalola and Shapiro, 1990; Kozłowska et
al., 2013) and could be due to luteinization spontaneously beginning in
non-ovulating, cystic follicles. However, the absence of any difference
between the serum concentrations of P4 in the serum of cyst-bearing and
non-cyst-bearing sows confirms that the hormonal abnormalities in the
follicular cysts are not reflected in the serum profiles of this steroid
(Babalola and Shapiro, 1990). It is interesting that the concentration of
P4 was positively correlated with BMP-15 concentration in the serum of
sows without follicular cysts. Based on this finding, I propose that the
interactions between BMP-15 and progesterone are involved in the control
of folliculogenesis in sows.
Thyroid hormones have also been suggested as participating in
folliculogenesis and/or the formation of follicular cysts (Fitko et al.,
1996; Błaszczyk et al., 2006; Stankiewicz et al., 2008). I tested the
concentration of FT4, which is a more reliable indicator of thyroid
status than total thyroxine concentration (Nowak, 1983). Here, I found no
differences between the concentration of FT4 in the serum of
cyst-bearing and non-cyst-bearing sows, and the recorded concentration of
this hormone in the serum of the examined sows was similar to concentrations observed
in pigs (Spiegel et al., 1993). Thus, neither the non-cyst-bearing nor the cyst-bearing sows in this study displayed hypo- or hyperthyroidism. In
contrast, Fitko et al. (1995) reported that the hypothyroid status of gilts
intensifies the cyst-formative actions of extrapituitary gonadotropins,
whereas hyperfunctioning of the thyroid significantly reduces this response.
The authors of that study also suggested that the mechanism of these
antagonist relations may be based on the interaction between receptors for
thyroid hormones and gonadotropins in the ovary and/or on the central or peripheral
relationship between thyroid hormone and estrogens (Fitko et al., 1995). The
results of this study indicate a possible local activity of thyroid hormones
during the formation of ovarian cysts because the concentration of FT4 was
significantly higher in the cysts than in preovulatory follicles. Moreover,
despite a similar concentration of FT4 in the serum of cyst-bearing and
non-cyst-bearing sows, I cannot exclude peripheral effects of thyroid
hormones in the pathogenesis of follicular cysts in pigs. In the present
study shown positive correlations between FT4 and BMP-15 and
GDF-9 in the serum of cyst-bearing sows have been. The participation of BMP-15 and
GDF-9 in the pathogenesis of follicular cysts in pigs has been suggested
(Stankiewicz and Błaszczyk, 2014, 2016). In addition, these factors,
depending on the stage of folliculogenesis, can either strengthen or weaken
the influence of gonadotropins on the ovarian follicle (Knight and Glister,
2006; Crawford et al., 2012). Also, the interactive effect of thyroid
hormones and gonadotropins on the formation of follicular cysts has been
shown in gilts (Fitko et al., 1995, 1996). Therefore, the relationships
between BMP-15, GDF-9, and FT4 in cyst-bearing sows may be associated
with a control of gonadotropins. This is a possible pathway for the action of
BMP-15, GDF-9, and FT4 in the mechanism of the formation of follicular cysts
in sows.
Conclusion
The data presented here will be useful for investigations into the potential
roles of the transforming growth factor β, thyroxine, and progesterone
during the formation of follicular cysts in sows.
The original data are available upon request from the corresponding author.
The author declares that he has no conflict of interest.
Edited by: M. Mielenz Reviewed by: two anonymous
referees
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