Deficiency in proliferative, angiogenic, and LH receptors in the follicle wall: implications of season toward the anovulatory condition.

This study aimed to gain insight on the effect of different seasons of the year on the expression pattern of growth factor and hormone receptors involved in follicle development. A novel follicle wall biopsy technique was used to collect in vivo follicle wall layers (ie, granulosa, theca interna, and theca externa) and follicular fluid samples from growing dominant follicles, simultaneously and repeatedly, using the same mares during the spring anovulatory (SAN), spring ovulatory (SOV), summer (SU), and fall ovulatory (FOV) seasons. The immunofluorescent expression patterns of epidermal growth factor receptor (EGFR), Ki-67, vascular endothelial growth factor receptor (VEGFR), and LH receptor (LHR) were evaluated in each follicle wall layer, in addition to intrafollicular estradiol and nitric oxide (NO). Proliferative proteins (EGFR and Ki-67) were highly (P < 0.05-P < 0.001) expressed during the SOV season compared with the SAN and FOV seasons. Lower (P < 0.05-P < 0.001) expression of both proteins was observed during SU compared with the SOV season. The expression of VEGFR was greater (P < 0.05-P < 0.01) in the theca interna of dominant follicles during the SOV season compared with the SAN and SU seasons. Similarly, in the overall quantification, the VEGFR expression was greater (P < 0.001) during the SOV season compared with the SU and FOV seasons. A higher (P < 0.05) LHR expression was detected in the theca interna during the SOV season than the SAN season. Furthermore, a higher (P < 0.05-P < 0.001) expression of LHR was observed in the granulosa, theca interna, and in the overall quantification during the SOV season compared with the SU and FOV seasons. Intrafollicular NO concentration did not differ (P > 0.05) among different seasons of the year. The intrafollicular estradiol concentration was higher (P < 0.05) during the SU compared with the SAN season and higher (P < 0.05) during the FOV season compared with the SAN and SOV seasons. In conclusion, the synergistic effect of lower expression of proliferative protein, angiogenic, and LH receptors in at least some of the layers of the follicle wall seems to trigger dominant follicles toward the anovulation process during the spring and fall transitional seasons.

Seasonal variation in equine follicular fluid proteome.

BACKGROUND: Proteomic studies of follicular fluid (FF) exist for several species, including the horse; however, the seasonal influence on FF proteome has not been explored in livestock. The application of high-throughput proteomics of FF in horse has the potential to identify seasonal variations of proteins involved in follicle and oocyte growth. METHODS: This study (i) profiles the proteomes of equine FF collected from dominant growing follicles during the spring anovulatory season (SAN), and spring (SOV), summer (SUM), and fall (FOV) ovulatory seasons; and (ii) identifies season-dependent regulatory networks and associated key proteins. RESULTS: Regardless of season, a total of 90 proteins were identified in FF, corresponding to 63, 72, 69, and 78 proteins detected in the SAN, SOV, SUM, and FOV seasons, respectively. Fifty-two proteins were common to all seasons, a total of 13 were unique to either season, and 25 were shared between two seasons or more. Protein-to-protein interaction (PPI) analysis indicated the likely critical roles of plasminogen in the SAN season, the prothrombin/plasminogen combination in SUM, and plasminogen/complement C3 in both SOV and FOV seasons. The apolipoprotein A1 appeared crucial in all seasons. The present findings show that FF proteome of SUM differs from other seasons, with FF having high fluidity (low viscosity). CONCLUSIONS: The balance between the FF contents in prothrombin, plasminogen, and coagulation factor XII proteins favoring FF fluidity may be crucial at the peak of the ovulatory season (SUM) and may explain the reported lower incidence of hemorrhagic anovulatory follicles during the SUM season.

Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue.

Preservation of cellular integrity and its mechanisms after ovarian tissue cryopreservation (OTC) and in vitro culture (IVC) procedures are crucial aspects for the success of preservation and recovery of female fertility. This study aimed to evaluate the effects of two cryopreservation methods (slow-freezing, SF, and vitrification, VIT) on the equine ovarian tissue after 1, 3, and 7 days of IVC by assessing: (i) preantral follicle morphology and distribution of follicle classes; (ii) protein expression of markers of cell proliferation for EGFR and Ki-67; (iii) markers of apoptosis for Bax and Bcl-2; and (iv) DNA fragmentation. Percentages of normal primordial follicles were similar (P > 0.05) among SF-control, VIT-control, and fresh control groups. After 7 days of culture, VIT-IVC7 had a greater (P < 0.05) total percentage of normal preantral follicles when compared with SF-IVC7, but both had a lower (P < 0.05) percentage than fresh IVC7 group. Prior to and after 7 days of culture, expression of EGFR and Ki-67 were similar (P > 0.05) among fresh, SF, and VIT groups. After 7 days of culture, VIT had higher (P < 0.05) Bax expression than the fresh and SF tissues, but Bcl-2 was similar (P > 0.05) among groups. Prior to IVC, TUNEL signals were similar (P > 0.05) among groups; however, VIT-IVC7 had greater (P < 0.05) TUNEL signals when compared with the fresh IVC7 group. In conclusion, findings demonstrated: (i) similar efficiency between SF and VIT compared with fresh control to preserve morphologically normal follicles; and (ii) similar tissue functionality and cell proliferation capability after equine OTC by either SF and VIT methods following IVC for 7 days. The results herein presented shed light on equine fertility preservation programs using OTC techniques.

In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels.

BACKGROUND: In vivo studies involving molecular markers of the follicle wall associated with follicular fluid (FF) milieu are crucial for a better understanding of follicle dynamics. The inability to obtain in vivo samples of antral follicle wall (granulosa and theca cells) without jeopardizing ovarian function has restricted advancement in knowledge of folliculogenesis in several species. The purpose of this study in mares was to develop and validate a novel, minimally invasive in vivo technique for simultaneous collection of follicle wall biopsy (FWB) and FF samples, and repeated collection from the same individual, during different stages of antral follicle development. We hypothesized that the in vivo FWB technique provides samples that maintain the normal histological tissue structure of the follicle wall layers, offers sufficient material for various cellular and molecular techniques, and allows simultaneous retrieval of FF. METHODS: In Experiment 1 (ex vivo), each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from 10-, 20-, and 30-mm follicles were repeatedly and simultaneously obtained through transvaginal ultrasound-guided technique. RESULTS: In Experiment 1, the thickness of granulosa, theca interna, and theca externa layers was not influenced (P > 0.05) by the harvesting techniques. In Experiment 2, the overall recovery rates of FWB and FF samples were 97 and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size. The expression of luteinizing hormone receptor (LHR) was mostly confined in the theca interna layer, with the estradiol-related receptor alpha (ERRα) in the granulosa and theca interna layers. The 30-mm follicle group had greater (P < 0.05) LHR expression in the theca interna and ERRα in the granulosa layer compared to the other groups. The overall expression of LHR and ERRα, and the intrafollicular estradiol were higher (P < 0.05 - P < 0.0001) in the 30-mm follicle group. CONCLUSION: The in vivo technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function, and has the potential to be translated to other species, including humans.

Pre-ovulatory follicle affects corpus luteum diameter, blood flow, and progesterone production in mares.

Color Doppler ultrasonography was used to study the temporal relationships between pre-ovulatory follicle (POF) and corpus luteum (CL) diameter and blood flow, with systemic progesterone (P4) concentration during two transitional ovulatory seasons in mares. Variables of POF and CL/P4 were evaluated for 6days before and 17days after ovulation, respectively. Evaluations were performed during two consecutive estrous cycles in spring and fall seasons, and during the last estrous cycle of the season. There were significant correlations among POF and CL variables, and P4 concentration that ranged from 0.24 to 0.95, and among the ratios of different variables that ranged from 0.39 to 0.92. There were linear regressions (P<0.01-0.001) for all comparisons among different variables. The POF diameter before the first ovulation of the season was larger (P<0.05), and POF vascularity was less (P<0.05), than in the last estrous cycle during the season. The CL blood flow was less (P<0.01) during the last compared with first pre-ovulatory period of the season. The POF diameters were positively correlated (r=0.67) during the two pre-ovulatory periods of spring and fall. Results provide evidence that the POF affects CL diameter and blood flow, and subsequently P4 production, and that POF diameter is repeatable within the same individual during different seasons.

Effects of FSH addition to an enriched medium containing insulin and EGF after long-term culture on functionality of equine ovarian biopsy tissue.

The effect of FSH supplementation on an enriched cultured medium containing insulin (10 ng/mL) and EGF (50 ng/mL) was investigated on in vitro culture of equine ovarian biopsy tissue. Ovarian tissue fragments were collected from mares (n = 10) and distributed in the following treatments: noncultured control, cultured control, and cultured + FSH. Both treated groups were cultured for 7 or 15 days. The end points evaluated were: follicular morphology, estradiol levels in the culture medium, fluorescence intensity for TUNEL, EGFR and Ki-67 detection, and gene expression of GDF-9, BMP-15, and Cyclin-D2 in the ovarian tissue. After seven days of culture, medium supplemented with FSH had a similar (P > 0.05) percentage of morphologically normal follicles compared to the noncultured control group. Estradiol levels increased (P < 0.05) from Day 7 to Day 15 of culture for both treated groups. No difference (P > 0.05) was observed for TUNEL and EGFR intensity between the noncultured control group and the treated groups after 15 days of culture. Ki-67 intensity did not differ (P > 0.05) between treated groups after 15 days of culture, but decreased (P < 0.05) when compared with the noncultured control group. Similar (P > 0.05) mRNA expression for GDF-9, BMP-15, and Cyclin-D2 was observed among all treatments after 15 days of culture. In conclusion, an enriched medium supplemented or not with FSH was able to maintain the functionality of equine ovarian biopsy tissue after a long-term in vitro culture.

Equine ovarian tissue viability after cryopreservation and in vitro culture.

Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmentation. Vitrification with EG had the highest rate of morphologically normal preantral follicles, while DMSO had the lowest (76.1 ± 6.1% and 40.9 ± 14.8%, respectively; P < 0.05). In slow-freezing, despite that DMSO had the highest percentage of morphologically normal follicles (77.7 ± 5.8%), no difference among the CPAs was observed. Fluorescence intensity of EGFR and Ki-67 was greater when vitrification with EG was used. Regardless of the cryopreservation treatment, DMSO had the highest (P < 0.05) Bax/Bcl-2 ratio; however, DNA fragmentation was similar (P > 0.05) among treatments after thawing. After in vitro culture, the percentage of normal follicles was similar (P > 0.05) between slow-freezing and vitrification methods; however, vitrification had greater (P < 0.05) stromal cell density than slow-freezing. In summary, equine ovarian tissue was successfully cryopreserved, increasing the viability of the cells in the ovarian tissue after thawing when using DMSO and EG for slow-freezing and vitrification methods, respectively. Therefore, these results are relevant for fertility preservation programs.

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

Changes in intrafollicular growth factors and hormones were evaluated in vivo in postdeviation and impending ovulation follicles. Mares (n = 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.