Association among lipopolysaccharide, the transforming growth factor-beta superfamily, follicular growth, and transcription factors in spontaneous bovine ovarian cysts

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Association among lipopolysaccharide, the transforming growth factor –beta superfamily, follicular growth, and transcription factors in spontaneous bovine ovarian cysts

H.E. Çolakoglu

^a

, S. Küplülü

^a

, I.M. Polat

^b

, M. Pekcan

^c

, E. Özenç

^d

, C. Baklac ı

^e

, K. Seyrek-_Intas¸

^f

, A. Gümen

^f

, M.R. Vural

^a^,^*

aDepartment of Obstetrics and Gynecology, Ankara University, Faculty of Veterinary Medicine, Ankara, Turkey

bDepartment of Obstetrics and Gynecology, Kırıkkale University, Faculty of Veterinary Medicine, Kırıkkale, Turkey

cDepartment of Biochemistry, Ankara University, Faculty of Veterinary Medicine, Ankara, Turkey

dDepartment of Obstetrics and Gynecology, Afyon Kocatepe University, Faculty of Veterinary Medicine, Afyonkarahisar, Turkey

eField Veterinarian, Alaca Farm, Bursa, Turkey

fDepartment of Obstetrics and Gynecology, Bursa Uludag University, Faculty of Veterinary Medicine, Bursa, Turkey

a r t i c l e i n f o

Article history:

Received 7 March 2019

Received in revised form 7 September 2019 Accepted 9 September 2019

Keywords:

LPS Ovarian cyst TGF-bfamily Growth factor Transcriptional factor Metabolite-metabolic hormones

a b s t r a c t

The aim of this study was to investigate some of the growth and transcriptional factors originating from oocytes and granulosa cells in follicularfluid and to identify the relationships between the basic blood metabolite-metabolic hormones and intrafollicular lipopolysaccharide (LPS) concentrations. Thirty cows included in the study were allocated into 2 groups comprising 15 cows with healthy preovulatory follicles (cyclic cows) and 15 cows with confirmed cystic follicles. The ovaries and uteri of all cows were assessed by transrectal ultrasonographic examination. Blood serum samples were collected at 15, 25, 35, 45, and 55 d after calving for analysis of nonesterified fatty acids,b-hydroxybutyrate, insulin, glucose, IGF- I, ACTH, and cortisol. Ovaries and uteri were examined using transrectal ultrasound. Vaginal discharge was evaluated on the same days. Follicularfluid was also aspirated on days 35–55 from the healthy preovulatory follicles and cystic follicles using a transvaginal ovum pickup method. The densitometric levels of inhibin-a, growth and differentiation factor (GDF-9), bone morphogenetic protein (BMP-6), and GATA-4 and GATA-6 proteins were analyzed by the Western blotting technique; the concentrations of antimullerian hormone (AMH), IGF-I, estradiol-17 beta (E2), and progesterone (P4) were determined by ELISA; and the concentrations of LPS in the follicularfluid were measured by the Limulus amebocyte lysate test. The serum insulin, ACTH, and cortisol concentrations were higher in cystic cows than cyclic cows, but serum IGF-I concentrations were lower in cystic cows. The IGF-I concentrations of cystic follicularfluids were lower, whereas AMH levels were significantly greater than those of healthy preovulatory follicularfluids. The cystic follicles had significantly lower expression levels of GDF-9, BMP-6, GATA-4, and GATA-6; in contrast, inhibin-aexpression and LPS concentrations were significantly higher than in healthy preovulatory follicles. The proportion of pathologic vaginal discharge within 25 d postpartum in cystic cows were higher than in the cyclic group. In conclusion, it is suggested that intrafollicular dysregulation of the transforming growth factor–bsuperfamily, growth, and transcriptional factors is affected by high intrafollicular LPS concentrations and systemic metabolic changes and these distur- bances may be responsible for the generation of ovarian cysts.

* Corresponding author. Tel.: þ90 312 317 03 15/4341; fax: þ90 312 317 83 81.

E-mail address:vural@ankara.edu.tr(M.R. Vural).

Contents lists available atScienceDirect

Domestic Animal Endocrinology

j o u r n a l h o m e p a g e : w w w . j o u r n a l s . e l s e v i e r . c o m / d o m e s t i c - a n i m a l - e n d o c r i n o l o g y

https://doi.org/10.1016/j.domaniend.2019.106398

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1. Introduction

Ovarian cysts associated with disturbance of normal ovarian cyclicity cause reproductive failure in dairy cows and economic losses [1]. The incidence of ovarian cysts was reported as 5% to 30% [2] in early postpartum (pp) cows and is most commonly seen between 14 and 40 d pp [1]. Ovarian cysts were deﬁned as nonovulatory follicular structures measuring equal to or greater than 25 mm that persist for at least 10 d without the presence of a functional ovarian corpus luteum (CL) [3]. However, despite intensive studies, the exact mechanism of ovarian cyst formation is unclear, with a multifactorial etiology. Cystic ovarian follicles are thought to result from neuroendocrine dysfunction of the hypothalamic-pituitary-gonadal axis [4]. An aberrant preovulatory LH surge and altered steroid production play important roles in cyst formation [5]. The unresponsiveness of follicular cells to the LH surge and lower LH receptor numbers are among the important mechanisms underlying ovarian cyst formation [6]. Cellular and molecular changes in growing follicles may also contribute to anovulation and cyst formation [7]. These multiple factors affect oocytes and ovarian somatic cells during follicular development, regu- lating follicular cell proliferation, differentiation, and the sensitivity of follicular cells to gonadotropins and other hormones [8,9]. Locally produced regulatory factors and proteins comprise various members of the transforming growth factor b ^(TGF-b) superfamily including bone morphogenetic proteins (BMP-6, BMP-15), antimullerian hormone (AMH), growth and differentiation factor (GDF-9), inhibin-a, activin, IGF-IGF-II, and transcriptional factors (GATA-4 and GATA-6). It has been reported that TGF-b family members exhibit either promoting or blocking effects on ovarian follicle growth and differentiation [10].

Gram-negative bacterial infections of the uterus are also known to downregulate ovarian steroidogenic enzymes, resulting in accumulation of progesterone (P4) and reduction of estradiol-17 beta (E2) production. Lipopolysaccha- rides cause increased cortisol production from the adrenal gland [11] and inflammation in follicular granulosa cells [12]. It was reported that LPS was higher in follicularfluid [9] of cystic cows. Cows with high follicular LPS concentrations also had higher P4 and lower E2 concentrations in follicularfluid [13]. In addition, it was reported that plasma cortisol and insulin concentrations increased and insulin resistance developed as a systemic response to LPS challenge [14]. It has also been established that cortisol acting as an anti-inflammatory agent can inhibit ovulation, with subsequent development and persistence of follicular cysts [15]. In dairy cows, both high and low concentrations of ACTH, insulin and IGF-I, and several metabolites of glucose, nonesterified fatty acids (NEFAs), andb-hydroxybutyrate (BHB) may all cause dysregulation of ovarian functions, finally leading to cyst formation [16].

New studies are needed to elucidate the exact mechanism of bovine ovarian cysts. In this study, we hypothesized that ovarian functions in cows would be associated with TGF-bsuperfamily proteins as well as metabolic hormones and LPS. Based on the aforementioned, the aim of the study was to investigate changes in TGF-bfamily proteins (GDF-9, BMP-6, inhibin-a, and AMH), IGF-I, and transcription factors

(GATA-4 and GATA-6), and to identify serum metabolite- metabolic hormone levels (NEFAs, BHB, insulin, glucose, IGF-I, ACTH, and cortisol) and to correlate these with follicularﬂuid LPS concentrations to understand the etio- pathogenesis of generation of follicular cysts.

2. Materials and methods

2.1. Sample collection and study design

This study was performed on a Holstein dairy cow herd.

Cows with high milk yield and a healthy periparturient period were housed in a free stall resting barn and fed a balanced ration according to age and yield characteristics.

The 320 cows in this study were examined for physiologic and pathologic ovarian cycles at 10-d interval from pp day 15 until pp day 55 d. Selected cows (n¼ 30) were assigned to the control or study group according to the presence of healthy preovulatory follicles or cystic follicles. Fifteen cows with cystic follicles at least 2.5 cm in diameter persisting for at least 10 d in the absence of a CL were included in the cystic group [17]. Another 15 cows with healthy preovulatory follicles 1.5–2.0 cm in diameter, and with uterine tissues that were highly turgid, contractile, and included transparent cervicalﬂuid were used as cyclic cows at 45–55 d pp [18]. Clinical signs of estrus were monitored twice daily. Ultrasonographic and gynecological examina- tions were performed at 10-d intervals for detection of CLs in the cyclic group.

The ovaries and uteri in all cows were assessed by transrectal ultrasonographic examination at 15, 25, 35, 45, and 55 d after calving. The healthy preovulatory follicular and cystic follicular diameters were recorded. At the same time, vaginoscopy was performed in both groups and vaginal discharges were evaluated [19].

2.2. Serum biochemical analyses

Blood samples were collected from the jugular vein at least 2 h after feeding to determine the levels of insulin, ACTH, cortisol, IGF-I, glucose, BHB, and NEFAs at 10- d intervals from days 15–55 after parturition. After blood collections, serum were separated by centrifugation for 10 min at 3,000 g and stored at 20C until assayed. In each serum sample, the concentrations of glucose (439– 90,901), NEFAs (34,691), BHB (417–73,501) were measured with colorimetric methods (WAKO). The levels of LH (CSB- E12826B), E2 (CSB-E13987B), P4 (CSB-E008172b), ACTH (CSB-E14322B), cortisol (CSB-E13064B), and IGF-I (CSB- E08893b) were analyzed using ELISA commercial reagent kits (Cusabio, Hubei, China).

2.3. Follicularﬂuid aspiration

Follicularﬂuid was aspirated from healthy preovulatory follicles using the transvaginal ovum pickup method 18 h after the onset of estrus which occurred at 45–55 d pp.

Follicularﬂuid in the cystic group was also aspirated with the same method at 35–45 d pp. Follicular puncture was performed using a disposable needle with an oocyte pick-up attachment mounted on a vaginal convex probe with a 135- 2

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degree angle of rotation [20]. Blood and tissue debris in the aspirated ﬂuids were separated by centrifugation at 3,000 rpm, and follicularﬂuids were stored at 20C until analyzed.

2.4. Electrophoresis, Western blotting, and hormone assays in follicularﬂuid

Growth and differentiation factor–9, GATA-4, GATA-6, BMP-6, IGF-I, and inhibin-alevels in follicularﬂuids were determined by the semiquantitative Western blotting technique. Proteins were separated in 10% SDS-PAGE. The same volume of follicularﬂuid sample was applied to each well. After electrophoresis, proteins were transferred to nitrocellulose membranes and blocked with 3% bovine al- bumin in TBST. Goat polyclonal IgG anti-mouse GATA-4, goat polyclonal IgG anti-human GATA-6, GDF-9, BMP-6, and inhibin-a(Santa Cruz Biotech., CA) were used as a primary antibody and bovine anti-goat IgG-HRP (Santa Cruz Biotech) was used as a secondary antibody. Proteins were detected by Luminol Reagent (Santa Cruz Biotech). Protein bands were scanned using a Bio-Rad GS-800 densitometer and signal intensity was determined with Quantity One Software (Bio- Rad) to compare expression levels among groups. Three independent experimental replicates were carried out.

The IGF-I (Cusabio, CSB-E08893b), P4 (GenWay, GWB- 3C719E) E2 (Cayman, 582251), and AMH (Beckman Coulter, A733818) concentrations in follicularﬂuids were determined by enzyme immunoassay-ELISA commercial reagent kits according to the manufacturers’ instructions.

Standard curve ranges were 6.6–4,000 pg/mL for E2, 0.2–

20 ng/mL for P4, 6.25–400 ng/mL for IGF-I and 0.16–

22.5 ng/mL for AMH. The respective intra-assay and inter- assay coefﬁcients of variations were 7.4% and 10.4% for E2, 10.7% and 12.5% for P4, 8% and 10% for IGF-1, and 2.9% and 4.9% for AMH.

2.5. Lipopolysaccharide measurement in follicularﬂuid

The presence of LPS in follicularﬂuid was detected using the QCL-1000 Chromogenic Limulus Amebocyte Lysate assay kit (Sigma E-TOXATE, ET0300) according to the man- ufacturer’s instructions [21]. Lipopolysaccharide concentrations were quantiﬁed by titration using E. coli standard dilutions. Standard curves of endotoxin ranges were 0.0015– 0.5 endotoxin units per milliliter (1 EU¼ 100 pg/mL).

2.6. Hormone analysis in blood serum

The levels of LH, E2, and P4 in serum samples were detected at 6-h intervals in cystic cows at pp 35–45 d. For determination of serum LH, P4, and E2 levels, three blood samples were taken at 6-h intervals after the onset of estrus in cyclic cows. Follicularﬂuids were also aspirated at 18 h after estrus.

2.7. Statistical analysis

The statistical package for social sciences was used for analysis of data (SPSS v14.1; Chicago, IL). Data were checked for equality of variance using Levene’s test. The Shapiro-

Wilk test was performed for normality of original and logarithmic values. When the distribution of values was not normal, data for serum biochemistry and hormone parameters were log-transformed before statistical analyses.

Student’s t-test was used when data were normally distributed, and the Mann-Whitney U test was used when data were not normally distributed. Student’s t-test was used to evaluate differences between the cystic and cyclic groups according to serum biochemistry and hormone parameters at each sampling time. Descriptive statistics for each variable were calculated and presented as the“mean

SEM.” Probability values of P < 0.05 were considered statistically signiﬁcant.

3. Results

3.1. Serum metabolic proﬁles

Serum glucose levels were not different among groups (excluding day 35 pp). However, cows with cystic follicles were hyperinsulinemic with significantly higher insulin concentration (greater than 50 mIU/mL) at days 15, 25, and 35 pp (P < 0.01) and had lower IGF-I concentration (P< 0.01) at days 35 and 45 compared with cyclic cows (Fig. 1). In cystic cows, the serum concentration of ACTH was significantly higher than cyclic cows at days 15, 25, 35, 45, and 55 pp (P< 0.01). Cortisol levels in cystic cows were also significantly greater (P < 0.05) at days 25 and 35 than in the cyclic group. The mean serum concentrations of NEFAs and BHB in both groups were similar and within normal reference ranges during the study period (Fig. 1).

3.2. Hormone and lipopolysaccharide concentrations in follicularﬂuid

The E2 and P4 concentrations in cysticfluids did not differ from those in follicular fluid of cyclic cows (P > 0.05; Table 1). The high concentration of E2 (>100 ng/mL) and the low level of P4 (<100 ng/mL) in cystic follicularfluid, similar to those levels in the healthy preovulatory follicles, suggest that these cystic follicles were estradiol-dominant cysts. Although the IGF-1 concentration in cystic follicularfluids was lower (P < 0.01), AMH levels were significantly greater (P < 0.01) than those of healthy preovulatory follicularfluids. The cystic cows had a significantly higher LPS concentration in their follicular fluid and also higher numbers of LPS-positive samples than cyclic cows (Table 1).

3.3. Expression levels of TGF-bsuperfamily proteins and transcriptional factors in follicularﬂuids

The molecular weights of inhibin-a, GDF-9, BMP-6, GATA-4, and GATA-6 in follicularfluids of cystic and cyclic cows were determined as 38, 51, 54, 44, and 56 KDa, respectively. The optical densities of these proteins are presented inTable 2. The follicularfluid of cystic follicles had significantly lower expression levels of GDF-9, BMP-6, GATA-4, and GATA-6; in contrast, inhibin-aexpression was significantly higher than in healthy preovulatory follicles (P< 0.01).

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3.4. Luteinizing hormone, E2, and P4 concentrations in serum

The mean LH concentration in serum of cyclic cows was higher than cystic cows at the time of follicular aspiration (P< 0.05) and at 6 h after aspiration (P < 0.05). Cyclic cows

had higher serum E2 concentrations at the time of follicle aspiration compared with cystic cows (P< 0.05). However, E2 concentrations were not signiﬁcantly different (P > 0.05, Table 3) 6 and 12 h after aspiration. In cystic cows, the level of circulating P4 was higher than cyclic cows at the time of 45

50 55 60 65 70

Glucose (mg/dL)

500 600 700 800

BHB ( μ mol/L)

10 20 30 40 50 60 70 80

Insulin (mIU/mL)

15 25

Po

5 35

*

35

5 35

ostpartum da

*

**

45 55

Cystic cows Cyclic cows

45 55

ays Cystic cows Cyclic cows

**

0.20 0.30 0.40 0.50 0.60 0.70

1

NEF A (mmol/L)

50 100 150 200 250

IGF-I (ng/ml)

s s

100 150 200 250 300 350

Cortisol (pg/mL)

5 25

15 25

Post

*

35 4

C C

35 4

C C

**

35 45

tpartum days C

*

C

45 55

**

5 55 s

0 2 4 6 8 10 12 14 16

15 25 35 45 55

ACTH (pg/mL)

Postpartum days Cystic cows Cyclic cows

**

Fig. 1. Least squares means (SEM) for glucose, NEFA, BHB, IGF-I, insulin, cortisol, and ACTH serum concentrations from day 15 to day 55 pp for cystic cows and cyclic cows (*P < 0.05, **P < 0.01). NEFA, nonesteriﬁed fatty acids; BHB,b-hydroxybutyrate; pp, postpartum.

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follicle aspiration (P < 0.05), 6 (P < 0.05), and 12 h (P< 0.05) after aspiration (Table 3).

3.5. Evaluation of follicle diameters and vaginal discharge in cystic cows

At day 15 d pp, dominant follicles developing into ovarian follicular cysts had greater diameters (mean diameter 1.58 0.28 cm) than healthy preovulatory follicles (mean diameter 1.26 0.31 cm; P < 0.01). The mean diameter of cystic follicles at days 25 and 35 pp were determined as 2.34 0.24 and 3.72 0.26 cm, respectively.

Twelve of the 15 cystic cows resumed cyclicity after follicular aspiration at day 35. The other 3 animals had recur- rence within 10 d after theﬁrst aspiration.

The proportion of cows characterized by purulent- mucopurulent vaginal discharge within 25 d pp in cystic cows (9 of 15) was higher than in the cyclic group (1 of 15).

These discharges changed into an intense mucolytic vaginal discharge after day 25 pp.

4. Discussion

This present study did notﬁnd signiﬁcant differences in NEFA and BHB concentrations between the two groups, whereas lower IGF-I concentration and higher ACTH and cortisol levels were found in cystic vs cyclic cows. Blood insulin concentrations in cystic cows were also higher than 48 mIU/mL (1.66 ng/mL) at days 15, 25, and 35 pp [22]. It was observed that a mucopurulent vaginal discharge continued until day 25 pp in cystic cows and their intrafollicular LPS concentration was quite high. In addition, cystic cows had lower serum E2 and LH concentrations and the blood P4 level was, interestingly, greater than 1 ng/mL in cystic cows at aspiration time compared with cyclic cows (0.40 0.07 ng/

mL). However, at 12 h after follicle aspiration, the serum LH and E2 levels were indistinguishable in the 2 groups.

Cows with endometritis had 2.43 times more ovarian cysts [3]. The carryover effect of uterine infections may disturb the ovulation mechanism by high intrafollicular LPS

levels or by systemic endotoxemia associated with ovarian inflammation [23]. Shimizu et al [6] demonstrated that high LPS in the follicularfluid of E2-active and E2-inactive cystic follicles is associated with low LH receptor expression in granulosa and theca cells. Lipopolysaccharide leads to overexpression of some cytokines (TNF alpha, IL-6, and IL- 10) from granulosa and theca cells. These factors also cause reduction in theca cell androstenedione production and decreasing E2 production in granulosa cells. Irregular E2 production blocks the LH surge, and this results in anovulation and cyst formation [24,25]. In the present study, cystic cows had higher LPS in follicularfluid, and the proportion of purulent-mucopurulent and intensive mucolytic vaginal discharge was higher in cystic cows at 15 and 25 pp.

Therefore, we suggest that higher LPS concentrations in follicularﬂuid occur as a result of uterine infections.

There is a linkage among inflammation, insulin insensi- tivity, and high plasma cortisol concentration through activation of the hypothalamic-pituitary-adrenal axis by proinflammatory cytokines stimulation. Furthermore, this stimulation decreases insulin-mediated glucose uptake and causes insulin resistance [26]. Blood insulin and glucose levels in cattle increase because of prolonged use of dexa- methasone [27]. Intramammary LPS challenge induces development of a systemic insulin resistance with concom- itant increase of adrenal cortisol and gluconeogenesis [12]. In the present study, we observed increased levels of ACTH and cortisol coexistent with higher blood insulin and lower blood IGF-I concentrations in cystic cows vs cyclic cows. In contrast to previous findings, although we observed high cortisol concentrations in cystic cows, these animals had a constant glucose level 30 d pp and a normal NEFA concentration, which may be related to peripheral glucose utilization from inflammatory and antilipolytic effects of LPS [28].

Glucocorticoids can regulate the activity of gonadotropins and steroid biosynthesis in the ovary. Increased cortisol concentrations due to ACTH, concurrent with hyperinsulinemia, reduce mRNA expression of LHr, 3b^-HSD, P450arom, and P450c17 and causes the formation of persistent follicles or cysts [15,29]. Findings in the present Table 1

Hormone concentrations and lipopolysaccharide endotoxin levels in cystic and healthy preovulatory follicularﬂuids (values are the mean SEM).

Groups Hormonal proﬁles of follicular ﬂuid Lipopolysaccharide endotoxin

levels (pg/mL)

E2(ng/mL) P4(ng/mL) AMH (ng/mL) IGF-I (ng/mL)

Cystic cows (n¼ 15) 226.46 57.2 40.4 11.2 61.46 27.0 20.24 4.1 6.69 0.95

Cyclic cows (n¼ 15) 255.92 63.1 28.38 22.3 9.06 7.0 30.29 3.1 1.36 0.30

P Value >0.05 >0.05 <0.01 <0.01 <0.01

Abbreviation: AMH, antimullerian hormone.

Table 2

Expression levels of proteins in the follicularﬂuids (optical density and values are the mean SEM).

Groups Expression levels of TGF-bsuperfamily proteins in follicularﬂuid

Inhibin-a GATA-4 GATA-6 BMP-6 GDF-9

Cystic cows (n¼ 15) 1.04 0.1 0.21 0.03 0.19 0.02 0.50 0.07 0.42 0.09

Cyclic cows (n¼ 15) 0.66 0.11 0.37 0.07 0.39 0.04 0.69 0.09 0.73 0.11

P Value <0.01 <0.01 <0.01 <0.01 <0.01

Abbreviations: TGF-b, transforming growth factor–b; BMP, bone morphogenetic protein; GDF, growth and differentiation factor.

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study suggest that cystic cows are adversely affected by high ACTH levels and elevated cortisol concentrations, compared with cycling cows. Presumably, the increase in proinﬂammatory cytokines resulting from clinical metritis and clinical endometritis may be induced by the hypothalamus-pituitary-adrenal gland axis and hence increases blood cortisol levels. Consequently, high blood cortisol may be responsible for hyperinsulinemia.

As is well known, the adrenal gland secretes several steroids, including hormones such as P4. As previously observed in response to ACTH challenge, blood P4 concentrations were increased up to intermediate levels in cystic cows (2.6 ng/mL) together with elevation of cortisol without CL formation; The adrenal gland was the source of P4 [17]. In agreement with these authors, it can be assumed that in the present study, the elevated serum P4 concentration (2.48 ng/

mL) without CL was related to chronic ACTH stimulation.

This intermediate concentration of P4 may cause the high frequency pulsatile release of LH or suppress the preovulatory LH surge, thereby inducing prolonged and excessive growth of the pp ﬁrst dominant follicle or resulting in transformation of the dominant follicle to a resistant form and to anovulation (like a vaginal sponge effect).

Blood IGF-I and insulin concentrations are metabolic indicators of healthy ovarian functions in cows. These metabolic hormones are also the basic hormones that provide metabolic regulation of ovarian functions [30]. In- sulin and IGF-I are associated not only with increasing LH receptor levels in granulosa and theca cells but also thereby with enhancing the steroidogenic response to gonado- tropin impulses [31,32]. In this study, we found that until pp day 45, blood insulin concentrations in cystic cows were greater than 48 mIU/mL, a condition known as hyperinsulinemia [22]. The high insulin concentrations and lower IGF-I levels may cause the decrease in E2 production, reduction of LH pulse intervals, and preovulatory LH surge deﬁciency because of decreasing cholesterol biosynthesis in ovarian follicular theca cells [33]. Elevated insulin levels can decrease serum sex hormone–binding globulin by affecting the hepatic production [26] and causing dysregulation of ovarian functions [34]. Insulin signaling is inﬂuenced by the responsiveness of its target tissues and the upregulation of insulin receptors (IRs) in the preovulatory follicle [35]. Insulin receptor upregulation is an important factor for follicular development because increased IR expression in preovulatory follicles is associated with increased E2 production.

The normal increase in blood insulin together with elevation of blood IGF-I during theﬁrst pp follicular wave resulted in increased circulating E2, associated with a reduced ratio of testosterone to E2, which occurred inde- pendently of any change in LH pulsatility [36]. On the other hand, Cheong et al [31] reported that cows which failed to ovulate theﬁrst pp dominant follicle are characterized by lower energy balance, increased insulin resistance, lower LH pulsatility, and lower intrafollicular concentrations of androstenedione and E2. In the present study, consistent with that of Cheong et al [31], cystic cows had lower IGF-I, lower LH, and E2 concentrations together with hyperinsulinemia compared with cyclic cows at pp days 35 and 45.

The follicularfluid reflects both biochemical and endo- crinological activity of the follicle [37]. Cysts with E2 concentrations>100 ng/mL in follicular fluid have been defined as estradiol-dominant cysts [38]. In this study, follicular fluid E2 concentrations were higher than 100 ng/mL, and all cysts were identified as estradiol-dominant cysts.

Physiologically, IGF-I together with insulin and BMPs, especially BMP-6, at the ovarian level regulate follicular selection, granulosa-theca cell differentiation, and proliferation at the early stage of follicular development [39], increase the sensitivity of follicles to the action of FSH and LH during the terminal stage of growth and development, and contribute to steroidogenesis and oocyte maturation [40]. The IGF-I is influenced by the action of IGFBPs and by the decreased level of IGF-I mRNA during cystic ovarian disease. Decreased IGF-I levels in follicularfluid affects the follicle response to FSH and LH and thereby abnormalities of ovarian steroidogenesis occur [41]. This study indicated that the follicularfluid IGF-I levels in E2-dominant type I cystic follicles were lower than in preovulatory follicles, similar to reports by Braw-Tal et al [38] and Rodriguez et al [41]. However, a previous study [9] demonstrated that IGF-I concentrations in P4-dominant type II cystic follicles which are also called “late-term cystic condition,” were higher than in preovulatory follicles in abattoir-derived ovaries.

Although this discrepancy between type I and type II cystic follicles is not understood, high IGF-I concentrations in type II cyst follicularﬂuid may arise from the activation of theca cells or breakdown of granulosa cells.

The AMH is expressed by granulosa cells and plays an important role in folliculogenesis, which is to control the formation of primary follicles thereby inhibiting excessive follicular recruitment by FSH [42]. The concentration of AMH in follicular ﬂuid is increased in women with Table 3

The blood serum concentrations of LH, E2, and P4 at different times after follicular aspiration in cystic and cyclic cows (values are the mean SEM).

Hormone Time Groups P value

Cystic cows (n¼ 15) Cyclic cows (n¼ 15)

LH (mIU/mL) Aspiration time 13.9 4.57 16.8 2.61 <0.05

6 h after aspiration 15.1 4.34 18.01 3.42 <0.05

12 h after aspiration 9.1 4.06 10.8 4.0 >0.05

E2 (pg/mL) Aspiration time 0.13 0.04 0.17 0.04 <0.05

P4 (ng/mL) Aspiration time 2.48 0.25 0.40 0.07 <0.05

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polycystic ovary syndrome (PCOS) [43] and in cows with cystic ovarian disease [9] and in follicles persisting for 10 and 15 d [2]. Similarly, the present study showed that AMH concentrations were signiﬁcantly higher in estradiol- dominant cystic follicles compared with cyclic preovulatory follicles. Overproduction of AMH in PCOS in women could be correlated with abnormally elevated insulin, LH, androgens, and oxidation products. The abnormal increase in AMH diminishes aromatase expression and follicular sensitivity to FSH leading to anovulation [44]. However, the exact mechanism of the association between hyperinsulinemia and an abnormal, AMH-dominant microenvironment was not elucidated in the present study, unlike PCOS cases in women. Further research is required into this subject. In this study, serum LH and serum E2 levels in cystic cows were not signiﬁcantly different from cyclic cows 12 h after aspiration and most of the cystic cows after cyst aspiration become pregnant. It is possible that the decreasing local production of AMH after aspiration may be linked to increased follicular responsiveness to FSH and release from follicular arrest, as seen after laparoscopic ovarian drilling in human PCOS. In human medicine, also, abnormally elevated AMH concentration in the blood is decreased after vitamin D3 supplementation, which may be attributed to the AMH gene promoter [44]. High intrafollicular AMH concentration and low BMP-6 expression were related to defects in growing follicles, especially in bovine-assisted reproduction in a previous study [9]. In contrast, Monniaux et al [7] reported that intrafollicular AMH concentrations in large follicles and cysts were not different and intrafollicular AMH was not a marker of cystic development in cows.

Bone morphogenetic protein–15 and GDF-9 are oocyte- specific growth factors playing key roles in granulosa cell development and fertility in animals [45]. Bone morphogenetic protein–15 and GDF-9 enhance or decrease the influence of gonadotropins on the ovarian follicle during its development [46,47] and synergistically affect the proliferation and differentiation of somatic cells, steroidogenesis, ovulation, and luteinization [45,47]. A study by Yan et al [48] demonstrated that knockout mice lacking GDF-9 and BMP-15 lost oocytes and exhibited ovarian cysts. In the present study, cystic follicular fluid had lower expression levels of GDF-9 than preovulatory follicles as shown using the Western blot technique. It is possible that reduction in GDF-9 expression is associated with oocyte degeneration depending on LPS or oocyte aging. Conflicting data on GDF- 9 concentrations in porcine cystic follicularfluid were reported by Stankiewicz [49] who showed that GDF-9 concentrations were higher than preovulatory follicles using ELISA technique on abattoir samples.

Previous studies [50,51] showed that GDF-9 expression levels in mature oocytes decrease in cows with intramammary-administered LPS. However, in line with previous knowledge, it is possible that synergistic BMP-15 and GDF-9 and other oocyte-specific growth factors such as GATA-4 and -6 may also be affected by LPS in cows. The data obtained in the present study indicated that oocyte- specific growth factors would be decreased in cows with high LPS in follicularfluid. It is thought that intrafollicular LPS may also affect the follicular microenvironment and

cause a decrease in growth factors derived from oocytes and mural cells.

Cystic follicles are not static structures and exhibit a cycle of degeneration followed by formation of new cysts.

The turnover interval for ovarian follicular cysts in cows depends on secretion of inhibin from cystic follicles and its blocking growth of new small follicles by inhibition of FSH secretion [52]. Inhibin A plays an important role in the extension of interwave intervals by suppressing recruitment of a new cohort of follicles [53]. The expression of inhibin-ain ovarian follicles is controlled by GDF-9 during early folliculogenesis and it acts as an antagonist of GDF-9.

Inhibin uses coreceptors with members of the TGF-b^family during follicular activity [54]. Binding of inhibin to these receptors may block the functions of other BMPs and GDF- 9. In GDF-9 null mice, inhibin-aexpression is elevated [55].

In this study, it was determined that cystic follicularfluid had higher inhibin-a expression levels accompanied by higher AMH concentrations and lower IGF-I levels than in preovulatory follicular fluids. These findings agree with studies showing that low GDF-9 in follicularfluids with high AMH and inhibin-aexpression in women with PCOS [56] are associated with developmental pathology of follicles. Physiologically, high GDF-9 expression and low AMH in the terminal stage of growth and development of follicles suppress inhibin-aexpression and thereby result in oocyte maturation [57].

The transcriptional factors GATA-4 and GATA-6 are expressed in granulosa and theca cells of growing follicles and regulate the expression of genes involved in folliculogenesis, oogenesis, and steroidogenesis. In mice, deficiency of GATA-4 and GATA-6 is associated with anovulation, inhibition of folliculogenesis, and a decrease in follicle- stimulating hormone receptor expression in granulosa cells [58]. The present study indicates that the expression levels of GATA-4 and GATA-6 are lower in E2-dominant cysts than in preovulatory follicles. This result agrees with thefind- ings of Polat et al [9] who reported that P4-dominant cystic follicles had lower GATA-4 and -6 expression. Thesefind- ings suggest that the lower expression of GDF-9, BMP-6, GATA-4, and GATA-6 in cystic intrafollicularfluid would be related to oocyte inactivation or the destructive effect of LPS on either somatic cells or oocytes.

5. Conclusion

In conclusion, results from this study showed that clinical metritis and clinical endometritis could impair the first pp ovulation mechanism via high intrafollicular LPS concentration and by triggering hypercortisolemia- hyperinsulinemia, and decreasing the IGF-I concentration in blood, it would lead to cystic ovarian disease in dairy cows. Furthermore, the components of follicularfluid were altered with lower IGF-I concentrations and with lower GDF-9, BMP-6, GATA-4, and GATA-6 expression levels. The present study also indicated that the high AMH and high inhibin-a expression in follicular fluid of E2-dominant ovarian cysts may be associated with defects in growing follicles and could serve as markers of oocyte competence in bovine-assisted reproduction.

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CRediT authorship contribution statement

H.E. Çolakoglu: Methodology, Investigation, Writing - review & editing, Writing - original draft. S. Küplülü:

Methodology, Investigation, Writing - review & editing, Writing - original draft. I.M. Polat: Methodology, Investi- gation, Writing - review & editing, Writing - original draft.

M. Pekcan: Methodology, Investigation, Writing - review &

editing, Writing - original draft. E. Özenç: Methodology, Writing - original draft. C. Baklacı: Methodology, Writing - original draft. K. Seyrek-_Intas¸: Writing - original draft. A.

Gümen: Writing - original draft. M.R. Vural: Methodology, Investigation, Writing - review & editing, Writing - original draft.

Acknowledgments

This work was supported by the Scientiﬁc and Techno- logical Research Council of Turkey, Turkey (TUBITAK) (Project number: 1090643).

The authors would like to thank Dr Barry Bavister for his excellent language editing, proofreading, and scientiﬁc critique of the article, and also Nurhan Dogan and Ersan Aloglu for statistical analyses.

There are no conﬂicts of interest for any of the authors.

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Association among lipopolysaccharide, the transforming growth factor-beta superfamily, follicular growth, and transcription factors in spontaneous bovine ovarian cysts