Changes in intrafollicular concentrations of free IGF-1, activin A, inhibin A, VEGF, estradiol, and prolactin before ovulation in mares

Theriogenology xxx (2016) 1–8

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Changes in intrafollicular concentrations of free IGF-1, activin A, inhibin A, VEGF, estradiol, and prolactin before ovulation in mares S.T. Bashir a, G.M. Ishak a, M.O. Gastal a, J.F. Roser b, E.L. Gastal a, * a b

Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, Illinois, USA Department of Animal Science, University of California, Davis, California, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 July 2015 Received in revised form 11 January 2016 Accepted 11 January 2016

Changes in intrafollicular growth factors and hormones were evaluated in vivo in postdeviation and impending ovulation follicles. Mares (n ¼ 30) were randomly assigned to five experimental groups based on target diameters of 25, 30, 35, 40 mm, and impending signs of ovulation. Furthermore, data belonging to two or more proximal diameter groups that were not different were combined and regrouped for each factor separately. Follicular fluid-free insulin-like growth factor 1 was highest (P < 0.003) in 35-mm follicles, followed by the 40-mm and impending ovulation follicle group, and the 25- to 30-mm follicle group. However, concentrations of insulin-like growth factor binding protein 2 in follicular fluid did not differ (P > 0.05) among groups. Additionally, follicular fluid activin A tended (P < 0.06) to be higher in impending ovulation follicles when compared with the 25- to 40-mm follicle group. Concentrations of intrafollicular estradiol were higher (P < 0.0001) in 40-mm and impending ovulation follicles than in the other follicle groups. Follicular fluid concentrations of inhibin A and vascular endothelial growth factor were lower (P < 0.05) in the 40-mm and the impending ovulation follicle group when compared with the 25- to 35-mm follicle group. Systemic and intrafollicular prolactin levels were lower (P < 0.05) in the impending ovulation group when compared with the 25- to 40-mm follicle group. Prolactin concentrations were higher (P < 0.05) in the follicular fluid than in the plasma. The novel findings of this study, a decrease in intrafollicular-free insulin-like growth factor 1, inhibin A, vascular endothelial growth factor, and prolactin during the final stages of follicular growth, document for the first time the occurrence of dynamic changes among intrafollicular factors and hormones during the stages of follicle dominance and as ovulation approaches. Ó 2016 Elsevier Inc. All rights reserved.

Keywords: Intrafollicular growth factor Hormone Follicle deviation Impending ovulation Mare

1. Introduction In mares, although good progress has been made in the use of assisted reproductive technologies like superovulation, ovum pickup, embryo transfer, and intracytoplasmic sperm injections [1], more information is required to

* Corresponding author. Tel.: þ1 618 453 1774; fax: þ1 618 453 5231. E-mail address: [email protected] (E.L. Gastal). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2016.01.013

achieve higher success rates. Knowledge of basic physiological mechanisms underlying ovulation, follicle maturation, oocyte quality, and embryo production is vital for the successful application (clinical and/or translational) of advanced reproductive technologies in mares. Additionally, the knowledge obtained from studying ovarian reproductive physiology in mares can be vital in understanding the reproductive function and ovarian physiology in women. Mares and women have significant similarities in ovarian follicular dynamics [2]. Owing to the number of similarities,

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several studies have used or advocated the use of the mare as a model for study of ovarian function in women [3–9]. Therefore, studies of ovarian follicular dynamics in mares provide a dual benefit in terms of advancement of knowledge in ovarian function of the mare and women. A follicular ovulatory wave in mares develops during the latter half of an estrous cycle, during which a cohort of growing follicles emerges, and usually one follicle is selected to be dominant, which ovulates by the end of the estrous cycle [10]. The process of selection of a single dominant follicle is termed deviation [11]. During the process of deviation, the dominant follicle continues to grow, whereas the rest of the follicles from the cohort grow at a reduced rate and then regress. The future dominant follicle forms a characteristic anechoic layer under the granulosa cell layer that can differentiate it from the second largest follicle [12]. At deviation, the changes in follicle growth rate and formation of the anechoic layer are temporally associated with increase in intrafollicular estradiol and free insulin-like growth factor 1 (IGF-1), decrease in circulating FSH, and increase in LH [11–17]. In mares, postdeviation growth of the dominant follicle is attributed to LH [16]. The transition of the largest growing follicle to dominant follicle and its subsequent growth is associated with systemic FSH and LH and is also affected by intrafollicular factors. A dynamic interrelationship has been observed between follicle selection and intrafollicular factors, such as free IGF-1, insulin-like growth factor binding proteins (IGFBPs), IGFBP protease, activins, inhibins, vascular endothelial growth factor (VEGF), steroids, GH, and others [14,17–22]. Furthermore, intrafollicular factors, especially free IGF-1 has been shown to be involved in continued follicular development [17,20,23–25] and ovarian cyclicity [22,26,27] in mares. Insulin-like growth factor 1 stimulates the growth of ovarian follicles by granulosa cell proliferation and differentiation in later stages of follicular development [24]. Moreover, IGFBPs play a vital role in regulating the concentrations of free IGF-1. In mares, IGFBP-2 and IGFBP-3 and their proteases are the most important for controlling the levels of free IGF-1 [28,29]. In addition, it has been observed that prolactin (PRL) might be produced in the ovarian follicles [30], but its role in follicle development and ovulation is not clear in mares. Although intrafollicular factors have been extensively studied during the process of deviation, knowledge is still lacking concerning dominance and preovulatory (postdeviation) phases of the estrous cycle. Most studies on follicular fluid factors in mares have categorized the follicles into ranges of small, medium, and large diameters [11,14,17,18,20,21,23,25]. However, none of the studies have compared changes in intrafollicular factors and hormones during different stages (diameters) postdeviation and in impending ovulatory follicles. Therefore, information about intrafollicular growth factors and hormones during postdeviation growth is critical for understanding the ovulatory and postovulatory physiological mechanisms, such as oocyte maturation, ovulation, and embryo development and survival that have important in vivo and in vitro applications. It is evident from recent studies that intrafollicular factors are important in the success of several assisted

reproductive technologies such as superovulation and embryo production [1,7,31]. Additionally, recent studies advocate the use of follicular fluid biomarkers for identifying quality of oocytes [32] to produce superior results, that is, production of offspring. A good intrafollicular environment is important for proper development and health of oocytes in several species, such as cattle [33], horses [28,34,35], and humans [36]. Therefore, it is important to properly understand the follicular microenvironment to comprehend the practical implications of different factors present in follicular fluid. This knowledge can be helpful in understanding follicular physiology and can also be exploited to generate better outcomes in assisted reproductive technologies. This study aimed to identify the dynamics of some of the important intrafollicular growth factors and hormones involved in the growth of dominant and preovulatory follicles from follicle deviation until ovulation. Identification of these factors will help in designing more targeted experiments in the future. 2. Materials and methods 2.1. Animals Mares (n ¼ 30) were used during an ovulatory season (April to October) in the northern hemisphere and handled in accordance with the US Department of Agriculture Guide for Care and Use of Agricultural Animals in Research. The mares were large ponies, 2 to 20 years (9.8  0.9 years), weighed 300 to 400 kg, and had docile temperament with normal reproductive tract as determined by ultrasonographic examination. Mares were kept under natural light in pasture with free access to water and trace mineralized salt. 2.2. Ultrasonographic examinations and groups Transrectal ultrasonographic examinations were performed after Day 12 of the estrous cycle (Day 0 ¼ ovulation) until a growing dominant follicle reached 25 mm in diameter. Subsequently, mares were randomly assigned into groups based on target diameters of 25 (n ¼ 6), 30 (n ¼ 5), 35 (n ¼ 5), 40 mm (n ¼ 5), and follicles that showed signs of impending ovulation (n ¼ 9) as described [37]. The largest follicle was measured on each day of examination. 2.3. Collection of follicular fluid and blood samples Follicular fluid was collected by ultrasound-guided transvaginal follicle aspiration [14,38] when the dominant follicle reached the target diameters. The aspirated follicular fluid was processed immediately in a refrigerated centrifuge (1500  g for 10 minutes), and the supernatant was stored at 20  C until hormone assays were performed. When the dominant follicle reached the target diameter, a single blood sample was collected from the jugular vein into heparinized tubes and immediately placed in ice water bath and processed in a refrigerated centrifuge (1500  g for 10 minutes), decanted, and stored (20  C) until assay.

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2.4. Hormone assays

3. Results

Concentrations of free IGF-1 were determined by a sandwich-type ELISA (Diagnostic Systems Laboratories, Webster, TX, USA); the interassay and intra-assay CVs were 4.4% and 6.1%, and the assay sensitivity was 0.05 ng/mL, respectively. A double-antibody radioimmunoassay (RIA) kit (Diagnostic Systems Laboratories) was used to quantify the IGFBP-2 concentrations; the intra-assay CV was 1.4%, and the assay sensitivity was 1.0 ng/mL. Follicular fluid estradiol was assayed by means of a double-antibody RIA kit (Diagnostic Products Corporation, Los Angeles, CA, USA; [14]) without extraction, and the samples were diluted 1:6000 in assay buffer (PBS with 0.1% gelatin). Intra-assay CV and assay sensitivity were 4.9% and 0.4 ng/mL, respectively. Concentrations of inhibin A were measured by solidphase sandwich ELISAs (Oxford Bio-Innovation Ltd., Oxfordshire, UK); follicular fluid samples were diluted to 1:1000 in fetal calf serum. Intra-assay and interassay CVs were 5.0% and 2.5%, and assay sensitivity was 1.0 ng/mL. Activin A concentrations were measured by a solid-phase sandwich ELISA (Oxford Bio-Innovation Ltd.); a sample dilution of 1:250 in 5% BSA was used. Interassay and intraassay CVs were 14.9% and 4.1%, and the assay sensitivity was 0.04 ng/mL. All the aforementioned hormone assays have been used previously for mare follicular fluid samples and had minimal cross reactivity with other hormones [18]. Concentrations of VEGF in the follicular fluid were determined using a competitive ELISA kit (Neogen Corporation, Lexington, KY, USA) from which an assay has been validated for use in mare follicular fluid [17]. The intraassay and interassay CVs for VEGF assay were 6.1% and 14.5%, and the sensitivity was 0.5 ng/mL. Plasma and follicular PRL concentrations were determined using a modified homologous double-antibody RIA as previously described [39]. The intra-assay and interassay CVs were 6.0% and 10.0%, respectively, with a sensitivity of 0.25 ng/mL.

3.1. Intrafollicular-free IGF-1 and IGFBP-2

2.5. Statistical analyses Data were tested for normal distribution using Shapiro– Wilk test and were transformed to ranks if not normally distributed. Outliers were identified using Dixon’s test and excluded from any further statistical analyses. One-way ANOVA was used to compare the data from multiple groups, and t test was used in the case of two groups. To identify significant changes in follicular fluid factors from deviation to preovulatory stage that were not apparent when the first overall analyses were done, the data were reassigned into new groups separately for each factor. Two or more adjacent groups not showing a difference were combined and compared in overall and discrete analyses. All the statistical analyses were performed using the SAS statistical software (version 9.2; SAS Institute, Inc., Cary, NC, USA). Systemic and follicular fluid PRL were compared using paired t test, and correlation between them was tested using Pearson’s correlation coefficient. A probability of 0.05 indicated that a difference was significant, and P > 0.05 or P < 0.1 indicated that the results approached a significant difference.

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Follicular fluid concentrations of free IGF-1 were different (P < 0.02) among various follicle groups (Fig. 1A). The 25-mm follicles had lower (P < 0.05) free IGF-1 when compared with 35-mm and impending ovulation follicles. Similarly, 30-mm follicles had lower (P < 0.05) concentrations of free IGF-1 compared with 35-mm follicles; no other follicle groups showed any significant differences. When the data were regrouped, free IGF-1 concentration was lower (P < 0.05) in the 25- to 30-mm follicle group, compared with the other groups. Furthermore, follicles from the 40-mm þ impending ovulation group had lower (P < 0.05) concentrations of free IGF-1 compared with 35mm follicles (Fig. 1B). On the other hand, concentrations of IGFBP-2 did not differ among follicle groups (Fig. 1C, D). 3.2. Intrafollicular estradiol, inhibin A, activin A, and VEGF Intrafollicular estradiol concentrations increased (P < 0.0001) with an increase in follicle diameter (Fig. 2A, B). Estradiol concentrations were lowest (P < 0.05) in 25-mm follicles, followed by 30- and 35-mm follicles, and were highest in 40-mm and impending ovulation follicles, before or after combining similar groups. On the other hand, concentrations of inhibin A did not show any difference among original follicle groups (Fig. 3A). However, after regrouping analyses, concentrations of inhibin A were higher (P < 0.02) in the 25- to 35-mm follicle group when compared with the 40-mm þ impending ovulation follicle group (Fig. 3B). Similarly, concentrations of activin A did not differ among follicle groups (Fig. 4A); however, after regrouping, the concentrations of activin A tended (P < 0.06) to be higher in the impending ovulation group than the 25- to 40-mm follicle group (Fig. 4B). Vascular endothelial growth factor follicular fluid concentrations were not different among the different follicular groups (Fig. 3C). However, VEGF concentration in the 40mm þ impending ovulation follicle group was lower (P < 0.002) than in the 25- to 35-mm follicle group (Fig. 3D). 3.3. Systemic and intrafollicular PRL Systemic concentration of PRL was higher (P < 0.05) in 35-mm follicles compared with 25-mm and impending ovulation follicles (Fig. 4C). However, no difference (P > 0.05) was detected in PRL concentrations in follicular fluid of various follicle groups (Fig. 4C). Follicular fluid PRL was higher (P < 0.05) in 40-mm and impending ovulation follicles than concurrent systemic concentrations. Systemic and follicular fluid concentrations of PRL were higher (P < 0.05) in the 25- to 40-mm follicle group when compared with impending ovulation follicles (Fig. 4D). Also, intrafollicular concentrations of PRL were higher (P < 0.05) in the 25- to 40-mm and impending ovulation groups than the associated systemic concentrations. Furthermore, no significant correlation between plasma and follicular fluid PRL concentrations was observed.

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Intrafollicular IGF-1

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Fig. 1. Mean (SEM) intrafollicular concentrations of free IGF-1 and IGFBP-2 in (A, C) individual and (B, D) combined follicle groups in mares. Bars with different superscripts within an end point are different (P < 0.05). The probabilities for an overall group effect are shown. IGF-1, insulin-like growth factor 1; IGFBP-2, insulin-like growth factor binding protein 2.

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Fig. 2. Mean (SEM) intrafollicular concentrations of estradiol in (A) individual and (B) combined follicle groups in mares. Bars with different superscripts within an end point are different (P < 0.05). The probabilities for an overall group effect are shown.

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Intrafollicular Inhibin A

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Fig. 3. Mean (SEM) intrafollicular concentrations of inhibin A and VEGF in (A, C) individual and (B, D) combined follicle groups in mares. Bars with different superscripts within an end point are different (P < 0.05). The probabilities for an overall group effect are shown. VEGF, vascular endothelial growth factor.

4. Discussion Identification of novel and more contrasting dynamic relationships of intrafollicular growth factors and hormones than we had anticipated was discovered by the combination of different proximal follicle groups. The present findings will be essential in designing future studies to investigate the role of the reported follicular fluid factors and hormones in mechanisms of the establishment of follicle dominance and growth of the dominant follicle during the preovulatory and impending ovulation phases. Additionally, a previous study [18] showed that intrafollicular factors and hormones have more intricate differential dynamics rather than following similar or opposing trends. Therefore, it is important to consider each individual follicular fluid factor or hormone based on its fluctuations over time during follicular development. Free IGF-1 continued to increase with increasing follicle diameter from 25 to 35 mm. Afterward, a novel decrease was observed in follicular fluid-free IGF-1 during the final stages of follicle development and before ovulation. On the

other hand, the concentrations of IGFBP-2 did not differ between the postdeviation follicular development and the time of impending ovulation. Previous studies have reported the important role of free IGF-1 in follicle selection in mares and heifers [17–20,40]. Furthermore, free IGF-1 has been shown to be important throughout continued follicular development [23–25] and also in ovulatory follicles [22,26,27] in mares. In spite of being one of the most studied follicular fluid factors, the role of free IGF-1 in postdeviation follicles and during impending ovulation is not clear. A regression or cessation in growth of the preovulatory follicle has been observed 2 days before ovulation in mares [37,41–44]. The current findings of a decrease in concentrations of free IGF-1 in the 40-mm þ impending ovulation groups could be a plausible reason a preovulatory follicle ceases to grow or decreases in diameter before ovulation. Our observation of no change in follicular fluid IGFBP-2 is in agreement with a previous study [28]. In this study, intrafollicular inhibin A and VEGF concentrations were lower in 40-mm þ impending ovulation follicles. Inhibin A has been shown to modulate the

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Fig. 4. Mean (SEM) intrafollicular concentrations of activin A and systemic and intrafollicular concentrations of PRL in (A, C) individual and (B, D) combined follicle groups in mares. Bars with different small superscripts (a, b) within an end point are different (P < 0.05). Differences between systemic and intrafollicular PRL are indicated by different capital superscripts (A, B). The probabilities for an overall group effect are shown. #Indicates a tendency for statistical difference between follicle groups or between systemic and intrafollicular PRL. PRL, prolactin.

responsiveness of granulosa cells to gonadotropins and steroidogenesis in vitro [45,46]. In addition, VEGF has been responsible for increasing the vascularity of the follicles and the permeability of endothelial cells of the follicular vasculature [47]. Furthermore, reports have shown that IGF-1 in vitro in cattle [48] and in vivo in mares [20] was able to increase concentrations of inhibin A and VEGF. Therefore, it is possible that the decrease in free IGF-1 before ovulation might be responsible for the decrease in inhibin A and VEGF, but this potential mechanism needs further investigation. Hours before ovulation, an abrupt decrease in follicular wall blood flow is observed in the ovulatory follicles in mares [8,9,49,50]. Therefore, it is conceivable that both inhibin A and VEGF levels are decreased in ovulatory follicles before ovulation in mares. The concentration of activin A was higher in impending ovulation follicles compared with the 25- to 40-mm follicle group. In contrast to free IGF-1 and inhibin A and VEGF,

activin A increased in the final stages of follicular development. Follicular fluid activin A has been involved in granulosa cell proliferation and in the upregulation of FSH receptors, estradiol synthesis, granulosa cell LH receptor expression, and also in enhancing oocyte maturation (reviewed in Ref. [45]). Furthermore, IGF-1, when injected into predeviation follicles in vivo, increased the concentrations of activin A in mares and heifers [20]; however, no knowledge is available concerning how IGF-1 affects activin A in postdeviation follicles. Our novel findings indicated higher intrafollicular concentrations of activin A in impending ovulation follicles although free IGF-1, VEGF, and inhibin A concentrations were lower as ovulation approached. Furthermore, contrasting changes in systemic concentrations of inhibin B and activin A have been previously reported in cycling aging women [51]. Therefore, further investigation is required to elucidate the mechanisms involved in contrasting intrafollicular dynamic

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changes of activin A compared with other follicular fluid factors during the preovulatory period in mares. Follicular fluid estradiol concentrations were higher in 40-mm and impending ovulation follicle groups followed by the other follicle groups. Follicular fluid and systemic estradiol increase with increasing follicle size has been reported previously [14]. Moreover, IGF-1 and activin A can increase in vitro production of estradiol by granulosa cells (reviewed in Ref. [18]). Therefore, based on our findings, activin A is a potential factor involved in continued production of estradiol during the preovulatory period in mares. A daily decrease in systemic estradiol concentration has been reported preceding ovulation in mares [41,42,52,53]. Our results of follicular fluid estradiol concentrations did not show any decrease as ovulation approached. Therefore, potential mechanisms involved in the decrease in systemic estradiol concentration, although follicular fluid estradiol does not decrease before ovulation, are: a decrease in vascular perfusion of the preovulatory follicle wall, resulting in decreased permeability of intrafollicular estradiol into the systemic circulation; a decrease in follicle size, which reduces the total concentration of intrafollicular estradiol; or both. Plasma and follicular fluid PRL concentrations were lower in impending ovulation follicles when compared with all other follicle groups. Also, the mean follicular fluid concentration of PRL was greater than the mean systemic concentration when the groups were combined. No significant detectable changes in systemic concentrations of PRL during the estrous cycle have been reported in mares [54]. However, several reports have related the beginning of luteolysis with a rise in systemic concentrations of PRL in the mare [55–57]. Periovulatory systemic PRL surge has been reported in mares, but a significant variability among animals and between seasons was also observed [58]. Furthermore, exogenous administration of estradiol increased systemic concentrations of PRL in mares and geldings [59–61]. Therefore, in the present study, lower systemic PRL levels in the impending ovulation group might have been associated with decreasing systemic estradiol concentration as previously described in mares [41,42,52,53]. A recent report [30] has suggested an ovarian source for PRL in the mare, which might explain the higher concentrations of PRL observed in follicular fluid when compared with plasma. Prolactin plays an important role in oocyte development and normal ovulation and enhances in vitro oocyte maturation in rodents (reviewed in Ref. [62]). Because the role of PRL in follicle development and ovulation is not very well understood in mares, more studies need to be undertaken to ascertain the source and function of intrafollicular PRL. In conclusion, after initial increasing levels during follicle growth, lower concentrations of intrafollicular-free IGF-1, inhibin A, VEGF, and PRL have been reported herein for the first time during the final phase of follicular maturation in mares. These findings could have been associated with a physiological process of decrease or cessation in follicular growth before ovulation. Also, the association between increasing levels of intrafollicular activin A and estradiol suggests that activin A might also be responsible for continued production of estradiol in preovulatory

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follicles before ovulation. Furthermore, the higher concentrations of intrafollicular PRL compared with systemic levels need to be further understood. Overall, the elucidation of the role of follicular fluid factors and hormones and their interactions needs to be studied further to allow a better understanding of the ovulatory process in mares. This knowledge will potentially help to obtain better outcomes during the use of assisted reproductive technologies and will also help to understand mechanisms of ovulatory dysfunctions like the hemorrhagic anovulatory follicle/ luteinized unruptured follicle syndrome in the mare and perhaps in other species.

Acknowledgments The authors thank Dr M.A. Beg and Lil Sibley for technical assistance with hormonal analyses and for reviewing the manuscript, and Dr O.J. Ginther, University of Wisconsin, Madison, USA, for allowing us to use some of the data included in this publication.

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