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.
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.
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).
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.
The knowledge about ovarian reserve is essential to determine the reproductive potential and to improve the methods of fertility control for overpopulated species, such as white-tailed deer ...(Odocoileus virginianus). The goal of this study was to evaluate the effect of age on the female reproductive tract of white-tailed deer, focusing on ovarian features. Genital tracts from 8 prepubertal and 10 pubertal females were used to characterize the preantral follicle population and density, morphology, distribution of follicular classes; stromal cell density; and apoptosis in the ovary. In addition, uterus and ovary weights and dimensions were recorded; and the number and the size of antral follicles and corpus luteum in the ovary were quantified. Overall, fawns had a greater (P < 0.05) preantral follicle population, percentage of normal follicles, and preantral follicle density than does. The mean stromal cell density in ovaries of fawns and does differed among animals but not between age groups. The apoptotic signaling did not differ (P > 0.05) between the ovaries of fawns and does. However, apoptotic ovarian cells negatively (P < 0.001) affected the preantral follicle morphology and density, and conversely, a positive correlation was observed with stromal cell density. As expected, the uteri and ovaries were larger (P < 0.002) and heavier (P < 0.001) in does than in fawns. In conclusion, this study has shown, for the first time, the preantral follicle population and distribution of classes, rate of morphologically normal follicles, and density of preantral follicles and stromal cells in white- tailed deer. Therefore, the findings herein described lead to a better understanding of the white-tailed deer ovarian biology, facilitating the development of new methods of fertility control.
Abstract Spermatozoa deliver more than the paternal genome into the oocyte; they also carry remnant messenger RNA from spermatogenesis. The RNA profiles of spermatozoa from high-fertility and a ...low-fertility Holstein bulls were analysed using Affymetrix bovine genechips. A total of 415 transcripts out of approximately 24,000 were differentially detected in spermatozoa collected from both bulls (fold change ⩾2.0; P < 0.01). These transcripts were associated with different cellular functions and biological processes. Spermatozoa from high-fertility bulls contained higher concentrations of transcripts for membrane and extracellular space protein locations, while spermatozoa from the low-fertility bulls were deficient of transcripts for transcriptional and translational factors. Quantitative real-time PCR was used on three low-fertility and four high-fertility bulls to validate the microarray data. Two highly represented transcripts in the microarray analysis (protamine 1 and casein beta 2) were validated, as well as a third transcript (thrombospondin receptor CD36 molecule) that showed a lower concentration in low-fertility bulls. This study presents the global analysis of spermatozoa originating from bulls with opposite fertility. These results provide some specific transcripts in spermatozoa that could be associated with bull fertility. Male fertility, ability of the sperm to fertilize the oocyte and support early embryonic development, is one of the most important factors in mammalian reproduction, and it has a significant impact on human health. Fertility evaluation of the male is essential for understanding the causes of poor quality semen and its specific effects on fertility. This study demonstrates that in addition to providing half of the genomic material, spermatozoa also contribute transcripts to the oocyte. The spectrum of mRNA molecules found in spermatozoa of high and low fertility bulls suggests potential roles for a number of these spermatozoa transcripts in fertilization and early embryonic development in mammals.
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.
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.
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.
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.
The aims of this study were to evaluate (1) the survivability of white-tailed deer ovarian tissue after cryopreservation by slow-freezing (SF) and vitrification (VIT) techniques and in vitro culture ...(IVC) for up to 7 days, and (2) the effects of cryopreservation techniques on protein expression of proliferative and apoptotic markers of ovarian tissue pre- and post-in vitro culture. Ovaries (n = 14) of seven white-tailed deer fawns (<1.5 years old) were used. Ovarian cortexes were cut into fragments (2 × 2 × 0.5 mm) and split into nine treatment groups: (1) fresh noncultured control, (2) fresh-IVC 1 day, (3) fresh-IVC 7 days, (4) SF noncultured, (5) SF-IVC 1 day, (6) SF-IVC 7 days, (7) VIT noncultured, (8) VIT-IVC 1 day, and (9) VIT-IVC 7 days. Preantral follicle morphology, class distribution, and density; stromal cell density; EGFR, Ki-67, Bax, and Bcl-2 protein expression; and DNA fragmentation were assessed. Results showed that: (i) white-tailed deer fresh ovarian tissue can be cultured for up to 7 days, preserving the tissue integrity and 50% of morphologically normal preantral follicles; (ii) cryopreservation of white-tailed deer ovarian tissue by either slow-freezing or vitrification does not disrupt markers of proliferation and apoptosis after thawing; (iii) ovarian fragments cryopreserved by the vitrification method had greater follicle viability during in vitro culture than the slow-freezing method; and (iv) fragments cryopreserved by slow-freezing suffered apoptosis earlier than those preserved by vitrification. The findings herein reported advance knowledge towards development of adequate cryopreservation protocols for long-term banking programs for Cervidae species.
•Feasibility of cryopreserving deer ovarian tissue by slow-freezing and vitrification methods has been shown.•Preantral follicle morphology of fresh ovarian tissue has been preserved after 7 days in culture.•Vitrification method had greater follicle viability during in vitro culture than slow-freezing method.•Apoptosis occurred earlier in ovarian fragments cryopreserved by slow-freezing method.
Numerous studies have reported the importance of thyroid hormones on the development of later preantral and antral follicles, but their interactions with other hormones and effects in regulating ...early preantral follicle growth remain unclear. Here we investigated the in vitro effects of thyroxine combined with insulin on caprine preantral follicle survival and development. Sliced ovarian tissues were cultured for 1 or 7 days using 10 ng/mL (low) or 10 μg/mL (high) insulin in the presence of thyroxine at 0, 0.5, 1 or 2 μg/mL. Post-culture, we evaluated the follicular survival and development, assessed the expression of apoptotic-related genes (Bcl2/Bax) and receptors of insulin and thyroid hormones, and quantified the estradiol and reactive oxygen species (ROS) production levels. Follicular survival in low-insulin culture conditions was enhanced by the presence of 0.5 μg/mL thyroxine (P < 0.05) as compared to the thyroxine-free medium but remained similar to non-cultured control in the presence of 2 μg/mL (P > 0.05). Significantly higher ROS production was measured from Day 1 to Day 7 in low-insulin culture media containing 0.5 or 2 μg/mL thyroxine (P < 0.05). When compared to high insulin level, the presence of thyroxine in low insulin culture conditions yielded higher stromal cell density (P < 0.05), increased estradiol production on Day 1, and higher Bcl2/Bax ratio on Day 7. Cultures with high levels of both insulin and thyroxine led to follicles and oocytes with larger diameters (P < 0.05). The RNA transcript levels of insulin and thyroid receptors were reduced in the presence of high insulin cultures when compared to controls (non-cultured). In conclusion, the combination of low concentrations of insulin and thyroxine better maintained follicle survival, while high levels ensured better follicular development.
•Insulin and thyroxine combination in early preantral follicles.
The aim of this retrospective study was to compare the number of follicles, cumulus oocyte complexes (COCs) and cultured In Vitro Produced (IVP) embryos obtained from 1396 non-stimulated Ovum Pick-up ...(OPU) sessions on 81 donor animals in a twice weekly OPU scheme. Results were obtained from 640 sessions following FSH-LH superstimulation, on 112 donors subjected to OPU once every 2 weeks. The stimulation protocol started with the insertion of an ear implant containing 3 mg norgestomet (Crestar, Intervet, Belgium) 8 days before puncture (day -8). The dominant follicle was ablated by ultrasound-guided follicle puncture on day -6. On day -3 and day -2, cows were injected with FSH (Ovagen, ICP) twice daily (8 am to 8 pm), i.e. a total dose of 160 μg FSH and 40 μg LG per donor per stimulation cycle. Animals were punctured 48 h after the last FSH injection (day 0). Progesterone implants were removed the next day. Stimulated donor cows were treated with this protocol at 14-day intervals. Follicles were visualized with a Dynamic Imaging ultrasound scanner, equipped with a 6.5 MHz sectorial probe. Follicles were punctured with 55 cm long, 18 gauge needles at an aspiration pressure corresponding to a flow rate of 15 ml/min. Cumulus oocyte complexes were recovered and processed in a routine IVF set-up. Results demonstrate that, expressed per session, FSH stimulation prior to OPU increases production efficiency with significantly more follicles punctured and oocytes retrieved. However, when overall results during comparable 2-week periods are considered (four non-stimulated sessions vs one stimulated), more follicles are punctured and more oocytes are retrieved using the non-stimulated protocol. No significant differences in the number of cultured embryos could be detected, indicating that FSH/LH stimulation prior to OPU might have a positive effect on in vitro oocyte developmental competence as more embryos are cultured with less, presumably better-quality, oocytes.
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.
•In vitro culture (IVC) of equine ovarian biopsy tissue for 7 or 15 days.•Enriched medium supplemented or not with FSH sustained IVC for 15 days.•Apoptosis rate was similar to fresh control after IVC for 15 days in both treatments.•Ovarian tissue estradiol production and gene expression were preserved for 15 days.
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.
•This study demonstrated the feasibility of culturing in vitro cryopreserved equine ovarian tissue.•Slow-freezing and vitrification had similar efficiency in preserving the morphology of preantral follicles.•Expression of EGFR and Ki-67 proteins were preserved after cryopreservation and in vitro culture for 7 days.•Expression of Bax and Bcl-2 protein in cryopreserved tissue was similar to that of fresh tissue after culture.•DNA degradation in cryopreserved-thawed ovarian tissue was similar to that in fresh tissue.
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.
•Follicle wall biopsies of dominant follicles were obtained in vivo from the same mares during the spring anovulatory, spring ovulatory, summer, and fall seasons.•Proliferative proteins (EGFR and Ki-67) were highly expressed during the spring ovulatory season compared with transitional seasons.•Vascular endothelial growth factor receptor expression was greater during the spring ovulatory season compared with fall ovulatory season.•Greater LHR expression was detected in the theca interna during the spring ovulatory vs anovulatory season.•A synergistic effect of lower proliferative protein, angiogenic, and LH receptors expression seems to trigger dominant follicles toward the anovulation process.
PDF
