EFFECT OF SOMATOTROPIN AND THYROXINE ON THE IN VITRO DEVELOPMENT OF BOVINE PREANTRAL FOLLICLES

The aim of the study was to evaluate the effect of recombinant bovine somatotropin (rbST) and thyroxine (T4) on survival and growth of bovine preantral ovarian follicles (PAOF) cultured in vitro. Ovarian fragments were collected in local abattoirs and immediately fixed for classical histology and transmission electron microscopy (non-cultured control). The other fragments were then cultured in situ for seven days in minimum essential medium alone (MEM+ cultured control) or in the presence of 1,000 ng/mL rbST and 20 ng/mL T4, isolated or associated. After seven days, there was a reduction (P<0.05) in the percentage of normal follicles in MEM+ alone or with T4. In oocyte diameter, there was a reduction in MEM+ alone. There was no influence (P>0.01) of the medium used on the follicular diameter of the PAOF cultured for seven days. Ultrastructural analysis showed cell damage. In conclusion, the presence of rbST maintains the rate of morphologically normal follicles during the culture for seven days (observed by optical microscopy), but it does not exert beneficial effects on its ultrastructural integrity and oocyte and follicular growth.


Introduction
Preantral ovarian follicles (PAOF) comprise about 90% of all follicular population present in the mammalian ovary (1) .However, in the physiological process occurring in vivo, more than 99% of them undergo atresia (2) .Thus, the increase in the reproductive potential of mammalian females is related to the maximum use of these ovarian follicles, enabled by the application of biotechniques.In this context, the manipulation of oocytes enclosed in preantral ovarian follicles stands out (3) .
The follicular development process is dependent on hormones and growth factors that act mainly in oocytes and granulosa cells (GC), promoting its development (4) .Therefore, studies that aim to obtain a culture system that allows the maintenance of the viability of these follicles and their posterior development are necessary.
In cattle, the first studies related to in vitro culture of PAOF occurred in the decade of 1990 (5) , and the best result reported for this species so far was the formation of the antral cavity (6) .However, an adequate culture system able to promote the complete development of PAOF in domestic animals has not been developed yet (7) .
Among the hormones currently studied in animal reproduction is somatotropin (ST).This hormone exerts influence in the mechanisms related to follicular growth and maturation, as well as in the regulation of physiological processes of the animals (8) , acting through the synthesis of insulin-like growth factor 1 (IGF-1) and IGF binding proteins (IGFBP) (9) .
Another hormone that has been a target of research in this area is thyroxine, synthesized by the thyroid gland.Arunakumari et al. (10) reported the influence of this hormone in combination with FSH in the in vitro development of PAOF of goats, in which they verified meiotically competent oocytes.Despite its known influence in ovarian folliculogenesis (11) , its effect on the development of PAOF of domestic animals is still little elucidated.Thus, by observing rbST and T4 potential, this study aimed to evaluate the effect of these hormones, isolated or in the association, on the survival and growth of bovine PAOF cultured in situ.

Material and Methods
This research was approved by the Ethics Committee on the Use of Animals of the Federal University of Viçosa (CEUA/UFV, process number 08/2013).Pairs of ovaries (n=5) from bovine females were collected at local abattoirs.Immediately after collection, they were washed in alcohol 70% and then in minimum essential medium (MEM), supplemented with 100 μg/mL penicillin and 100 μg/mL streptomycin.Subsequently, they were transported to the animal reproduction laboratory (DVT/UFV) in MEM at 4 ºC for approximately one hour, according to Chaves (12) .
In the laboratory, they were placed in a Petri dish containing MEM with HEPES, on which fragments of approximately 3x3 mm (1 mm thick) were obtained using tweezers and scalpel blades under sterile conditions.A fragment from each ovary pair was randomly selected and immediately fixed for analysis by classical histology (CH) and transmission electron microscopy (TEM), constituting the non-cultured control.The other fragments were individually cultured in 1 mL medium for seven days, in a culture dish at 38 °C and 5% CO2.It is worth mentioning that each ovarian pair corresponded to a repetition and, therefore, five repetitions were performed.
The base medium, called MEM (pH 7.2-7.4),was supplemented with ITS (10 μg/mL insulin, 5.5 μg/mL transferrin, and 5 ng/mL selenium), 2 mM glutamine, 2 mM hypoxanthine, and 1.25 mg/mL bovine serum albumin (BSA).The culture performed only with the base medium supplemented was called cultured control (α-MEM + ), and the other treatments consisted of the culture with α-MEM + added of 1000 ng/mL recombinant bovine somatotropin (rbST) or 20 ng/mLthyroxine(T4), or added of both hormones (1,000 and 20 ng/mL, respectively).These concentrations were used after previous studies performed by our team when added to in vitro culture of PAOF of cattle (13) and goats (14) .The culture media were stabilized for one hour before use, being completely renewed every two days.
After seven days of culture, the fragments were fixed in Carnoy for four hours and directed to CH. Primarily, they were dehydrated at increasing concentrations of ethanol, being then diaphonized in xylol.The fragments were then embedded in paraffin blocks and serially sectioned to the thickness of 7 μm.Each section was placed on a slide and stained by the Periodic acid-Schiff technique and hematoxylin.All sections were examined under an optical microscope (Olympus), at 400x magnification.For each treatment, 30 follicles were counted, random without choice, degenerate or normal.
In relation to follicular survival, the follicles were classified as normal or degenerated.PAOFs present in the ovarian tissue were considered morphologically normal when they presented granulosa cells (GC) organized around the oocyte, whereas it was spherical or slightly elongated, with nucleus without evidence of pyknosis (15) .Degenerated follicles were characterized according to GC disorganization around the oocyte and to the presence of areas of cytoplasmic retraction and/or nuclear pyknosis (16) .
Oocyte and follicular diameters were measured with the aid of an ocular micrometer, and the measurement was performed on follicles classified as normal in the histological analysis.
Procedures for transmission electron microscopy (TEM) were performed according to Costa (17) .Small fragments (1 mm 3 ) of ovarian tissues were fixed for four hours at room temperature in Karnovsky (2.5% glutaraldehyde and 4% formaldehyde in 0.1 M cacodylate buffer, pH 7.2).Afterward, the samples were washed twice in 0.1 M cacodylate buffer, post-fixed in 1% osmium tetroxide, dehydrated in alcohol and embedded in Epon 812 resin (18) .Initially, semithin sections (0.5 μm) were performed with glass knives in ultramicrotome.These were stained with toluidine blue, placed on a slide/coverslip and examined under a light microscope, selecting the best semithin sections for the ultrathin sections (60-70 nm).The ultrathin sections were obtained with a diamond knife and collected on (200 mesh) copper grids, stained with uranyl acetate and lead citrate, according to the protocols of Watson (19) and Reynolds (20) .Subsequently, the ultrathin sections were examined by transmission electron microscopy as the integrity and density of the cytoplasmic organelles of the oocyte and GC, the degree of cytoplasmic vacuolization, and the integrity of cell membranes.
The variables were submitted to normality (Lilliefors) and homoscedasticity (Cochran) tests, followed by analysis of variance at a probability of 5%.In case of significance, the most appropriate test of comparison between means was performed, avoiding type I and II statistical errors.When they did not satisfy the requirements of normality and homoscedasticity, even after the appropriate transformations (angular for percentages and radicial for diameters) data were submitted to Kruskal-Wallis non-parametric test (21) .

Results and Discussion
Normal (Fig. 1: A and B) and degenerated (Fig. 1: C and D) follicles were observed in both groups (non-cultured and cultured).In morphologically normal follicles, the oocyte had a spherical or slightly elongated format, with organized GC nucleus without the presence of cytoplasmic retraction, as reported by Bruno et al. (15) .In contrast, degenerated follicles had retracted oocytes, with disorganized GC and the presence of nuclear pyknosis (16) .
After seven days of culture, there was a reduction (P<0.05) on the rate of normal follicles in fragments cultured in the absence of hormones (α-MEM + ) and in the medium containing T4, as shown in Table 1.This result shows that rbST was beneficial, once its presence in the medium maintained follicular viability during the culture for seven days.
The composition of the culture medium used as a base is one of the major factors that affect follicular survival and development rates (22) .In the present study, the medium used was MEM + , and some supplements were added to this medium, such as insulin, transferrin, selenium and bovine serum albumin.Insulin allows a greater use of the medium energy sources (23) .Selenium has the function of activating enzymes that participate in the process of eliminating free radicals produced by cells (24) .However, these substances were not able to maintain follicular viability rate, when compared to that observed in normal PAOF of the non-cultured group.
These results evidence the importance of rbST in the culture medium since both media containing only rbST or an association of rbST with T4 maintained (P>0.05)follicular survival during in vitro culture for seven days.
It is known that the major rbST action mechanism on folliculogenesis occurs by the induction of increased plasma and ovarian concentrations of IGF-1 (25) .This growth factor, in turn, is capable of suppressing DNA fragmentation by apoptosis, acting as an anti-apoptotic factor (26) .In cattle, some studies showed that GH acts through IGF-I (8) .This hormone increases the development of small antral follicles at the gonadotropin-dependent stages and stimulates oocyte maturation, while IGF increases proliferation of GC, steroidogenesis and oocyte growth in the majority of mammal species (27) .Thus, this condition could justify the maintenance of PAOF normality when cultured in medium containing rbST, as verified in the present experiment.
In relation to the role of T4, some authors reported beneficial effects in animal folliculogenesis (10) and in the culture of PAOF of goats (14) .Serakides et al. (28) verified that rats treated with free T4 showed maintenance of follicular viability.Thyroid hormones have beneficial effects on the differentiation of follicular cells (28,29) .Furthermore, Serakides et al. (28) observed a positive and significant correlation between primary follicles and T4 plasma concentrations, indicating that there is the participation of thyroid hormones on the development of primordial follicles, with consequent formation of primary follicles.However, the contribution of this hormone for follicular survival was not evidenced in the present study.According to Dijkstra et al. (29) and Jiang et al. (30) , low T4 concentration can negatively affect folliculogenesis, leading to a greater number of atretic follicles.On the other hand, this condition had no influence on the present experiment, since the medium in the absence of T4, that is, only the base medium, also did not maintain the percentage of normal follicles during in vitro culture compared to the non-cultured control.
Regarding oocyte diameter, a reduction in the average diameter when PAOF were cultured in medium without the addition of rbST and T4 was verified (Table 2).However, this diameter was maintained after the cultures in medium containing these hormones, in isolated or associated manner.The the necessity of improvements in the composition of media for in situ culture of bovine PAOF.

Conclusions
Although the presence of rbST in culture medium of bovine preantral follicles maintained the rate of morphologically normal follicles during the culture for seven days (observed by optical microscopy), the use of rbST and T4 in in situ culture medium of bovine PAOF, at the concentrations used in the present experiment, does not exert beneficial effects on its ultrastructural integrity and oocyte and follicular growth.