Bovine Preantral Follicles Research Articles

Effect of somatotropin and thyroxine on the in vitro development of bovine preantral follicles

Effect of somatotropin and thyroxine on the in vitro development of bovine preantral follicles

Expression of TNF-α system members in bovine ovarian follicles and the effects of TNF-α or dexamethasone on preantral follicle survival, development and ultrastructure in vitro

This study was conducted to detect the protein expression of TNF-α system members (TNF-α/TNFR1/TNFR2) in bovine ovarian follicles and to evaluate the effects of TNF-α or dexamethasone on the survival and growth of primordial follicles in vitro, as well as on gene expression in cultured ovarian tissue. It was hypothesized that TNF-α induces follicular atresia in ovarian tissues cultured in vitro, and that dexamethasone suppresses the production of endogenous TNF-α, which can improve follicle viability in vitro. Ovarian fragments were cultured for 6days in α-MEM+ supplemented with TNF-α (0, 1, 10, 100 or 200ng/ml) or dexamethasone (0, 1, 10, 100 or 200ng/ml). After culture, the expression of mRNAs for BCL-2, BAX, P53, TNF-α, and CASP3 and CASP6 were evaluated. Immunohistochemical results showed that the TNF-α system members, were detected in bovine preantral and antral follicles. After 6days, the TNF-α (10ng/ml) treatment reduced the percentage of normal preantral follicles and increased the number of TUNEL-positive cells in cultured tissue. Dexamethasone (10ng/ml) during 6days of culture did maintain the percentage of normal follicles and the ultrastructure of follicles, while the presence of TNF-α or dexamethasone did not influence primordial follicle activation. However, TNF-α or dexamethasone had no effect on the levels of mRNA for P53, BCL-2, BAX and CASP6, in cultured tissues, but the presence of dexamethasone reduced the levels of CASP3 compared to ovarian slices cultured in control medium (α-MEM+). In conclusion, proteins of the TNF-α system are expressed at different bovine follicle stages. The addition of TNF-α in culture reduces follicle survival and increases the number of apoptotic cells in ovarian tissue, while the presence of dexamethasone maintains follicle ultrastructure in cultured tissue.

Ultrastructure of bovine preantral follicles: effect of the slaughterhouse aseptic process on follicular integrity

Veterinaria México OA ISSN: 2448-6760Cite this as:Cisneros Prado JL, Montiel Palacios F, Canseco Sedano R, Merchant Larios H. Ultrastructure of bovine preantral follicles: effect of the slaughterhouse aseptic process on follicular integrity. Veterinaria México OA. 2017;4(2). doi: 10.21753/vmoa.4.2.424.Ovaries from slaughtered cows are the most abundant source of preantral immature follicles for in vitro studies including maturation and in vitro fertilization. Integrity of immature follicles may be compromised by inadequate management during the collection and transport of the gonads, limiting the success of a correct evaluation. This study assessed the effect of aseptic and transport of bovine ovaries on the structural and ultrastructural integrity of immature follicles from the ovarian cortex. Ovaries from 20 different crossbred cows (Bos taurus × B. indicus) were randomly assigned to one of two aseptic and transport treatments: (1) the ovaries were washed once with 70 % ethanol for 10 s, rinsed twice with normal saline solution (OH + NSS), and then transported in NSS + 0.05 % gentamicin sulphate; or (2) the ovaries were washed three times with phosphate buffered saline (PBS) and transported in PBS + 0.05 % gentamicin sulphate. Samples of the ovarian cortex were obtained from each treatment group. Half of samples of each treatment was processed for histologic evaluation of follicular morphology and developmental stage using haematoxylin & eosin, and the other half was processed to analyse the ultrastructure of immature follicles using transmission electronic microscopy. The rates of good-quality follicles were 36.95 % and 88.6 % for OH + NSS and PBS, respectively. Histological analysis showed higher number of abnormal follicles in OH + NSS than in PBS treated ovaries (F1,12 = 99.64, P = 0.0001). The proportions of morphological alterations were not significantly different between treatments (χ21,7 = 7.72, P = 0.358). The ultrastructure of immature follicles from OH + NSS ovaries showed cell damage, revealed by abundant cell detritus around both, the oocyte and follicular cells. However, the presence of alterations was barely evident in both the cytoplasm and the nucleus of the granulosa cells.Figure 2. Semi-fine cuts of immature follicles present in bovine ovarian cortex treated with PBS (A, B, C; viewed at 40X) or OH+NSS (D, E, F; viewed at 40X).

Effects of bone morphogenetic protein 4 (BMP4) on in vitro development and survival of bovine preantral follicles enclosed in fragments ovarian tissue.

The aim of this study was to evaluate the effects of different concentrations of BMP4 on activation, development and mRNA expression of GDF9, BMP15, PCNA, Bax and Bcl2 in cultured bovine follicles enclosed in ovarian tissues. Ovarian tissue fragments were cultured for 6 days in α-MEM+ alone or supplemented with different concentrations of BMP4 (10, 50 or 100 ng/ml). Classical histology was performed to analyze follicle growth and morphology, while real-time PCR was used to analyze mRNA levels in fresh and cultured tissues. After 6 days, the culture of ovarian tissue in α-MEM+ alone or supplemented with 10, 50 or 100 ng/ml BMP4 promoted follicular activation. The different concentrations of BMP4 maintained the percentage of normal follicles similar to results of the control. The presence of 100 ng/ml BMP-4 in culture medium increased oocyte and follicular diameters of primary and secondary follicles when compared with those follicles from uncultured control or cultured in α-MEM+ alone (P < 0.05). The tissues cultured in the presence of increasing concentrations of BMP4 had an increase in mRNA expression of the tested genes, but despite this the differences were not statistically significant. In conclusion, 100 ng/ml BMP4 promotes an increase in diameters of follicles and oocytes of primary and secondary follicles after 6 days of in vitro culture.

Effects of Growth Hormone on In Situ Culture of Bovine Preantral Follicles are Dose Dependent.

The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (insitu) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross-breeds defined were cultured insitu for 1 and 7days in minimal essential medium (α-MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50ng/ml). The ovarian fragments non-cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey's and Dunnett's tests) and chi-square test (χ(2) ). After 7days of culture, the treatment with 50ng/ml GH showed no differences with fresh control (p>0.05) and had greater effectiveness than in the 0, 10 and 25ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50ng/ml GH. Follicles cultured in 10ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50ng/ml GH is the most effective concentration for the development of preantral follicles cultured insitu.

Effects of IGF‐1 on In Vitro Culture of Bovine Preantral Follicles are Dose‐Dependent

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF-1) in the development, survival and ultrastructure of the bovine preantral follicles cultured insitu. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7days in 1ml of α-MEM(+) , supplemented with different concentrations of human recombinant IGF-1 (0, 30, 70 and 100ng/ml), in an incubator at 37°C and 5% of CO2 in 24-well plates with total replacement of the medium every 2days. Non-cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF-1 30ng/ml treatment was similar to the fresh control (p>0.05) both on the day 1 and on the day 7 of invitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p>0.05). After 7days of culture, the IGF-1 30ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p<0.05). Also, after 7days of culture, IGF-1 30ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF-1 tested. Ultrastructurally, the non-cultured control and IGF-1 30ng/ml, after 7days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF-1 30ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured invitro.

Impact of insulin concentration and mode of FSH addition on the in vitro survival and development of isolated bovine preantral follicles

The insulin and FSH are two important substances in the folliculogenesis process. Thus, the hypothesis of this experiment is that insulin concentration and the form of FSH addition affect the in vitro survival, growth, and estradiol production after culture of isolated bovine preantral follicles. The effects of insulin concentration (experiment 1) and the influence of both fixed and sequential concentrations of FSH (experiment 2) on the in vitro survival and development of bovine preantral follicles were investigated in this study by IVC for 18 days. In experiment 1, on Day 18 of culture, the addition of insulin at all concentrations promoted follicular survival rates significantly higher than that of the control, with the 10-ng/mL insulin treatment showing values significantly higher than the other treatments. The addition of 5- and 10-ng/mL insulin promoted higher follicular growth than the control and other treatments. In experiment 2, FSH 100 had a higher percentage of follicular viability compared with the control. FSH 100 produced follicle diameters significantly higher than those of the control and FSH seq. treatment. Estradiol levels in the presence of FSH (fixed concentration) were significantly higher than the other treatments. In conclusion, the association of insulin (10 ng/mL) and fixed concentration FSH (100 ng/mL) provides high rates of survival, growth, and estradiol production in bovine preantral follicles.

Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus–oocyte complex

The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus–oocyte complexes (COCs) from antral follicles (>2–6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect in vitro culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC.

Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol

Abstract: The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.

Influence of BMP-2 on early follicular development and mRNA expression of oocyte specific genes in bovine preantral follicles cultured in vitro.

This study evaluates the effect of different concentrations (0, 10, 50 and 100ng/mL) of bone morphogenetic protein-2 (BMP-2) on primordial and secondary follicle development. It also investigates the effects of FSH and BMP-2 on the growth, morphology, ultrastructure and expression of mRNA for GDF9, NLRP5 and NPM2 genes in secondary follicles cultured for 18 days. The presence of BMP-2 at all tested concentrations increased the development of primordial follicles in vitro, but the highest concentration of BMP-2 (100 ng/mL) reduced the percentage of normal follicles when compared with tissues cultured with 10 ng/mL BMP-2. During culture of secondary follicles, in contrast to higher concentrations (50 or 100 ng/mL), 10 ng/mL BMP-2 kept the morphology of follicles during initial stages of in vitro culture. This concentration of BMP-2 also benefits maintenance of the ultrastructure of 18-day cultured follicles. The presence of both BMP-2 and FSH in culture medium resulted in a significant (P<0.05) increase in follicular diameter after 18 days of culture. However, both FSH and BMP-2 reduced follicular mRNA expression of GDF9 and NLRP5 when compared to follicles cultured in media containing only FSH. In combination with FSH, BMP-2 reduced the mRNA levels of NPM2, when compared to follicles cultured in control medium. It is concluded from these data that 10 ng/mL BMP-2 promotes the growth of primordial in vitro and it helps to maintain the ultrastructure of secondary follicles, while FSH is more important for better expression of follicular markers like GDF9 and NLRP5.

Long-term in vitro culture of bovine preantral follicles: Effect of base medium and medium replacement methods

Two culture media and replacement methods were compared during long-term in vitro culture of secondary follicles of cattle using α-MEM+ or TCM-199+ as base media. The medium replacement methods were: Conventional – removal and subsequent addition of the same amount (60μl) in a 100μl aliquot (MEM-C and TCM-C), and Small Supplementation – addition of 5μl of fresh medium to an initial small aliquot (50μl), resulting in a final volume of 125μl on the last day of culture (MEM-S and TCM-S). A total of 207 secondary follicles were cultured individually for 32 days at 38.5°C in 5% CO2 and medium replacement was performed every other day. The MEM-S treatment resulted in a larger (P<0.01) follicular diameter, greater (P<0.02) growth rate, greater (P<0.02) antrum formation, as well as greater (P<0.0001) estradiol concentrations when compared with the MEM-C treatment. The medium change methods did not affect (P>0.05) the follicular and estradiol end points for TCM-199+. The expression of the FSHR gene was greater (P<0.03) with the TCM-C than TCM-S treatment, while the relative amounts of mRNA for IGF1 was greater (P<0.02) with MEM-S than TCM-S treatments and for VEGF was greater (P<0.02) with MEM-C than TCM-C treatment. In conclusion, the type of base medium and the effect of periodic addition of medium differentially affected follicle development, estradiol production, and gene expression. Furthermore, α-MEM+ can be used to replace TCM-199+ for culture of preantral follicles of cattle if progressive addition of medium is used for medium change.

Effects of neutral red assisted viability assessment on the cryotolerance of isolated bovine preantral follicles

Fertility preservation strategies warrant non-invasive viability assessment of preantral follicles (PAF) such as staining with Neutral Red (NR) that is incorporated by viable follicles. To optimize the procedure, we firstly determined the lowest concentration and shortest exposure time needed for optimal viability screening of isolated bovine PAF. Secondly, we combined this protocol to a vitrification procedure to assess cryotolerance of the stained follicles. Isolated PAF (900, divided over 6 replicates) were cultured in DMEM/Ham's F12 (Culture Medium - Cm) for 4 days (38.5 °C, 5% CO2). On D0, D2 and D4, follicles were stained, by adding NR medium (NRm = Cm with different concentrations NR) after which viability was assessed by counting stained/non-stained PAF every 30 min for a period of 2 h. Following a binary logistic regression analysis with staining as a result (yes/no) versus log-concentration, a probability model could be fitted, indicating that the proportion of stained follicles remained stable after 30 min when 15 μg/ml NR was used, without compromising follicular health and viability. Consequently, using this protocol, no significant effect of staining prior to vitrification, was found on PAF viability immediately after warming or following 4 days of culture. In conclusion, we propose NR staining as a non-invasive, non-detrimental viability assessment tool for PAF, when applied at 15 μg/ml for 30 min, being perfectly compatible with PAF vitrification.

Frozen and Fresh Ovarian Tissue Require Different Culture Media to Promote in Vitro Development of Bovine Preantral Follicles

The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.

In vitro culture of bovine preantral follicles: a review.

Preantral follicles are the majority of the ovarian follicle population and their use as a source of homogeneous oocytes for bovine reproductive biotechnologies could result in a substantial advance in this field. However, while in other species embryos and offspring have been produced, in bovine species the results have been limited to the follicular activation of small (primordial) preantral follicles and formation of early antral follicles from large (secondary) preantral follicles after in vitro culture. Therefore, this review will highlight the basic aspects of bovine folliculogenesis by focusing on preantral follicles, the methods of harvesting preantral follicles, the main results from in vitro follicular culture during the last 20 years, and the potential candidate substances (basic supplements, growth factors, and hormones) for improving the efficiency of in vitro follicle growth.

In vitro development of bovine secondary follicles in two- and three-dimensional culture systems using vascular endothelial growth factor, insulin-like growth factor-1, and growth hormone

The aim of this study was to evaluate the development and estradiol production of isolated bovine secondary follicles in two-dimensional (2D, experiment 1) and three-dimensional (3D using alginate, experiment 2) long-term culture systems in the absence (control group; only α-MEM+) or presence of vascular endothelial growth factor (VEGF), insulin-like growth factor-1, or GH alone, or a combination of all. A total of 363 isolated secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO2 in a humidified incubator with addition of medium (5 μL) every other day. In 2D culture system, follicular growth and antrum formation rates were higher (P < 0.05) in VEGF treatment compared with the other treatments. In 3D culture system, only estradiol concentration was greater (P < 0.05) in the GH than in the control group, whereas the other end points were similar (P > 0.05). In summary, this study demonstrated that the benefits of using a certain type of medium supplement depended on the culture system (2D vs. 3D). Vascular endothelial growth factor was an effective supplement for the in vitro culture of bovine secondary follicles when the 2D culture system was used, whereas GH only affected estradiol production using the 3D culture system. This study sheds light on advancements in methodology to facilitate subsequent studies on bovine preantral follicle development.

Fresh and vitrified bovine preantral follicles have different nutritional requirements during in vitro culture

The aim of this study was to compare the efficiency of different media for the in vitro culturing of fresh and vitrified bovine ovarian tissues. Fragments of the ovarian cortex were subjected to vitrification and histological and viability analyses or were immediately cultured in vitro using the alfa minimum essential medium, McCoy's 5A medium (McCoy), or medium 199 (M199). Samples of different culture media were collected on days 1 (D1) and 5 (D5) for quantification of reactive oxygen species and for hormonal assays. In non-vitrified (i.e., fresh) ovarian tissue cultures, the percentage of morphologically normal follicles was significantly greater than that recorded for the other media (e.g., M199). In the case of previously vitrified tissues, the McCoy medium was significantly superior to the other media in preserving follicular morphology up until the last culture day (i.e., D5), thus maintaining a similar percentage from D1 to D5. Reactive oxygen species levels were higher in D1 vitrified cultured tissues, but there were no differences in the levels among the three media after 5days. The hormonal assays showed that in the case of previously vitrified tissues, at D5, progesterone levels increased on culture in the M199 medium and estradiol levels increased on culture in the McCoy medium. In conclusion, our results indicate that the use of M199 would be recommended for fresh tissue cultures and of McCoy for vitrified tissue cultures.

Differential effects of activin-A and FSH on growth, viability and messenger RNA expression in cultured bovine preantral follicles

This study evaluated the effect of follicle stimulating hormone (FSH) alone or in combination with activin-A on survival, growth and expression of mRNA for follicle stimulating hormone receptor (FSH-R), proliferating cell nuclear antigen (PCNA), activin receptor type I B (ActR-IB), activin receptor type II B (ActR-IIB), hyaluronan synthase-1 (HAS 1) and hyaluronan synthase-2 (HAS 2) in bovine secondary follicles cultured in vitro for 18 days. Preantral follicles (~0.2mm) were isolated and individually cultured in absence (α-minimum essential medium (α-MEM+): control) or presence of activin-A, FSH in increasing concentrations or both activin-A and FSH. An increase in follicular diameter was observed after 6 days of culture in all culture treatments compared to control medium; after 12 days in treatment with FSH alone or the mixture of FSH and activin-A (P<0.05); and, after 18 days only in the presence of FSH alone (P<0.05). However, in combination with activin-A, FSH-stimulated follicle growth after 18 days was blocked (P<0.05) and, had significantly lower (P<0.05) levels of mRNA for ActR-IB, ActR-IIB, when compared to α-MEM+, and for FSH-R and PCNA, when compared to α-MEM+ supplemented with activin-A only. Moreover, follicles cultured in presence of FSH alone had greater (P<0.05) levels of mRNA for HAS 1 and HAS 2 than those cultured in medium supplemented with both activin-A and FSH. Morphological, immunofluorescence and ultrastructural analysis confirmed the integrity of follicles cultured in FSH after 18 days. In conclusion, activin-A exerts a stimulatory effect on in vitro early follicular development for up to 6 days, has no effect after 12 days of culture and blocks the stimulatory growth effect of FSH after 18 days. The reduced mRNA levels in follicles cultured with both activin-A and FSH suggest a decreased sensitivity of follicles for activin-A and FSH and inhibited follicular cell proliferation after long-term in vitro culture of isolated preantral follicles.

Effect of fetal calf serum and bovine serum albumin on in vitro development of bovine preantral follicles during three-dimensional culture

Effect of fetal calf serum and bovine serum albumin on in vitro development of bovine preantral follicles during three-dimensional culture

Growth and antrum formation of bovine primary follicles in long-term culture in vitro

Successful antral formation in vitro from bovine preantral follicles (145–170μm) has been described previously, but antrum formation from the primary follicle (50–70μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50–70μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E2), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH+LH+E2+bFGF or FSH+LH+E2+bFGF+EGF (p<0.05). An addition of 50ng/ml bFGF or bFGF+25ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50ng/ml bFGF+25ng/ml EGF to media containing FSH+LH+E2 turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.

Comparative study on the in vitro development of caprine and bovine preantral follicles

The present study aimed to evaluate the effect of the addition of IGF-1 to the in vitro culture (IVC) medium on the development of caprine and bovine preantral follicles ≥150μm in diameter. Caprine and bovine preantral follicles were cultured for 18 days in the absence or presence of 50ng/mL IGF-1. Follicles were classified according to morphological characteristics, and those showing morphological signs of degeneration such as darkness of oocytes and surrounding cumulus cells or those with misshapen oocytes were classified as degenerated. Oocyte and follicular diameter were measured only in healthy follicles and antral cavity formation was defined as a visible translucent cavity within the granulosa cell layers. Regarding follicular growth, the daily increase in follicular diameter was calculated, and oocyte extrusion was determined by calculating the rates of ruptured basement membrane with the release of cumulus oocyte complexes (COCs). After IVC, oocytes were submitted to in vitro maturation (IVM) and morphological analysis using fluorescent markers. IGF-1 did not affect follicular morphology and antrum formation in both species. However, follicular extrusion and in vitro matured oocytes were obtained only in caprine. It can thus be said that although bovine and caprine preantral follicles react differently at same culture conditions, this study showed the successful in vitro development of caprine preantral follicles. We also suggest the enrichment of the IVC media and increase in the IVC period for bovine preantral follicles.