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 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.
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.
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.
The objective of this study was to determine whether (1) systemic and intrafollicular cortisol concentrations in horses are directly related and (2) supraphysiological levels of glucocorticoids ...affect in vitro maturation (IVM) rates of oocytes. Specifically, we studied the (1) changes in the intrafollicular cortisol and progesterone in context with granulosa cell gene expression during maturation of equine follicles (from 5–9 mm, 10–14 mm, 15–19 mm, 20–24 mm, and ≥25 mm in diameter) and (2) effects of cortisol supplementation on IVM rates and gene expression of equine cumulus–oocyte complexes (COCs). For these purposes, follicular fluid, granulosa cells, and COCs were collected from 12 mares (mean age 8.6 ± 0.5 yr) by transvaginal aspiration. Cortisol and progesterone concentrations in follicular fluid from follicles ≥25 mm were greater (P < 0.05) than in all other follicle classes and were positively correlated (r = 0.8; P < 0.001). Plasma concentrations of cortisol and progesterone did not differ before and after follicle aspiration (P > 0.05). In granulosa cells, gene expression of NR3C1, HSD11B1, HSD11B2, and CYP21A2 did not differ (P > 0.05) among different follicle classes. Maturation rates were similar (P > 0.05) among groups, regardless of the cortisol concentration in the IVM medium. In cumulus cells, messenger RNA expression of genes involved in glucocorticoid mechanism and apoptosis was either increased (NR3C1 and BCL2) or decreased (HSD11B2) by treatment (P < 0.01). In oocytes, gene expression of maturation markers (BMP15 and GDF9) was affected (P < 0.001) by cortisol treatment. This study demonstrates the involvement of glucocorticoids in follicle and oocyte maturation and cortisol modulation by HSD11B2 in equine COCs. Our data provide further information for understanding the normal ovarian endocrine physiology which might in turn also help improve equine assisted reproduction techniques.
•Cortisol is increased in follicular fluid from larger follicles.•Cortisol addition during in vitro maturation did not impair maturation rates.•Glucocorticoids are involved in follicle and oocyte maturation in horses.
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.
Changes in systemic concentrations of FSH, LH, oestradiol and progesterone during the ovulatory follicular wave were compared between 30 mares and 30 women. Based on a previous study, the emergence ...of the future ovulatory follicle was defined as occurring at 13.0 mm in mares and 6.0 mm in women, and deviation in diameter between the two largest follicles was expected to begin at 22.7 mm in mares and 10.3 mm in women. Mean FSH concentrations were high in mares during the luteal phase, resulting from statistically identified FSH surges occurring in individuals on different days and in different numbers (mean, 1.5 ± 0.2 surges/mare); the internadir interval was 3.9 ± 0.3 days. In contrast, mean FSH in women was low during the luteal phase and increased to a prolonged elevation during the follicular phase. The prolonged elevation was apparent in each individual (internadir interval, 15.2 ± 0.4 days). Changes in LH or oestradiol concentrations encompassing deviation were not detected in mares, but both hormones increased slightly but significantly between emergence and deviation in women. The hypothesis that a greater number of growing follicles causes a greater predeviation decrease in FSH was supported for mares (r, -0.39; P< 0.04), but a similar negative correlation (r, -0.36) was not significant in women. The hypothesis that the increase in oestradiol during the luteal phase in women was at least partly attributable to luteal-phase anovulatory follicular waves was not supported. Normalization of FSH concentrations to the day of emergence showed maximum value on the day of emergence with a significant increase and decrease on each side of emergence in both species. The day of expected deviation occurred 3 days after emergence during the decline in FSH in both species. These results indicated that the previously reported striking similarities in emergence and deviation between mares and women during the ovulatory follicular wave are associated with species similarities in the temporal relationships between follicle events and FSH concentration changes. Thus, mares may be useful research models for studying the role and mechanism of the action of FSH in emergence and deviation during the ovulatory follicular wave in women.
Deviation in growth rates of the follicles of the ovulatory wave begins at the end of a common growth phase and is characterized
by continued growth of the developing dominant follicle (F1) and ...regression of the largest subordinate follicle (F2). Follicle
diameters during an interovulatory interval were compared between 30 mares and 30 women, using similar methods for collecting
and analyzing data. Follicles were tracked and measured daily by ultrasonography. Diameter at follicle emergence (mares, 13
mm; women, 6 mm) and the required minimal attained diameter for assessment of follicles (mares, 17 mm; women, 8 mm) were chosen
to simulate the reported ratio between the two species in mean diameter of F1 at the beginning of deviation (mares, 22.5 mm;
women, 10.5 mm). F1 emerged before F2 ( P < 0.02) in each species, and the interval between emergence of the two follicles was similar (not significantly different)
between species. Growth rate for F1 and F2 during the common growth phase was similar within species, and the percentage of
diameter increase was similar between species. Proportionality between species in diameter of F1 at deviation (2.2 times larger
for mares than for women) and at maximum preovulatory diameter (2.1 times larger) indicated that relative growth of F1 after
deviation was similar between species. A predeviation follicle was identified in 33% of mares and 40% of women and was characterized
by growth to a diameter similar to F1 at deviation but with regression beginning an average of 1 day before the beginning
of deviation. The incidence of a major anovulatory wave preceding the ovulatory wave was not different between species (combined,
25%). Results indicated that mares and women have comparable follicle interrelationships during the ovulatory wave, including
1) emergence of F1 before F2, 2) similar length of intervals between sequential emergence of follicles within a wave, 3) similar
percentage growth of follicles during the common growth phase, and 4) similar relative diameter of F1 from the beginning of
deviation to ovulation. Similar follicle dynamics between mares and women indicate the mare may be a useful experimental model
for study of folliculogenesis in women, with the advantage of larger follicle size.
The functional and temporal relationships between circulating gonadotropins and ovarian hormones in mares during Days 7â27
(ovulation = Day 0) was studied using control, follicle ablation, and ...ovariectomy groups (n = 6 mares/group). In the follicle-ablation
group, all follicles â¥6 mm were ablated on Day 7, and every 2 days thereafter, newly emerging follicles were also ablated.
Estradiol concentrations decreased ( P < 0.01) similarly in the controls and the follicle-ablation group between Days 7 and 11 and by Day 15 began to increase in
the controls and continued to decrease in the follicle-ablation group. Concentrations of progesterone were not affected by
follicle ablation, but diameter of the corpus luteum was greater ( P < 0.05) by Day 21 in the follicle-ablation group; these results indicated that the follicles were involved in morphologic
luteolysis, but not in functional luteolysis. Concentrations of LH were higher ( P < 0.05) on Days 15 and 16 in the follicle-ablation group than in the controls, indicating an initial negative effect of follicles
on LH. Immunoreactive inhibin and estradiol decreased ( P < 0.0001) and FSH and LH increased ( P < 0.05) within 1 or 2 days after ovariectomy; these changes occurred more slowly in the follicle-ablation group. The maximum
value for an FSH surge in each control mare was below the lower 95% confidence limit in the ovariectomy group. Maximum concentration
for the periovulatory LH surge in the controls was not different from the mean maximum LH concentrations in the ovariectomy
group. Our interpretation is that the gonadotropin surges resulted from changes in the magnitude of the negative effects of
ovarian hormones on the positive effects of extraovarian control. There was no indication of a positive ovarian effect on
either FSH or LH.
Abstract
Surges in circulating gonadotropins reflect the negative effects of ovarian hormones acting to oppose the positive effects
of extraovarian control
Abstract
STUDY QUESTION
Do growth patterns and endocrine profiles differ between ovulatory follicles (OvFs) and luteinized unruptured follicles (LUFs) in women?
SUMMARY ANSWER
Growth rates, diameters ...and associated endocrine profiles differed between OvFs and LUFs in unstimulated cycles.
WHAT IS KNOWN ALREADY
Two-three waves of antral follicles develop during the menstrual cycle in ovulatory women of reproductive age, with the second or third wave terminating in ovulation. In contrast, some women can develop LUFs, where a preovulatory follicle fails to rupture and there is subsequent luteinization of the follicle wall. However, no study has compared OvFs and LUFs in unstimulated cycles.
STUDY DESIGN, SIZE, DURATION
This retrospective observational study was conducted in 56 healthy women of reproductive age (range: 19-41 years) and with a history of regular menstrual cycles.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Participants who met inclusion criteria were enrolled, as previously reported. Daily transvaginal ultrasonography was performed for one interovulatory interval (IOI) to measure the diameters of all follicles >2 mm. Blood samples were collected every 3 days during the IOI to measure serum concentrations of FSH, LH, estradiol and progesterone.
MAIN RESULTS AND THE ROLE OF CHANCE
The interval from emergence to deviation (i.e. follicle selection) was shorter (P < 0.05) for LUFs compared to OvFs. However, the intervals from emergence to maximum diameter and deviation to maximum diameter were longer (P < 0.05) for LUFs compared to OvFs. Follicle deviation in LUFs occurred at a larger diameter (P < 0.05) compared to OvFs, and LUFs grew to larger (P < 0.0001) diameters compared to OvFs. Moreover, LUFs grew faster (P < 0.05) from emergence to deviation and from deviation to maximum diameter, compared to OvFs. LUFs were associated with low (P < 0.05) systemic LH levels at emergence and maximum diameter compared to OvFs. LUFs were also associated with low (P < 0.05) systemic FSH and high (P < 0.05) systemic progesterone at deviation and maximum diameter, respectively. Estradiol was higher (P < 0.05) at deviation and lower (P < 0.05) at maximum diameter for LUFs compared to OvFs.
LIMITATIONS, REASONS FOR CAUTION
A 3-day interval of blood sampling for hormonal analyses was conducted, as a more frequent sampling interval was not considered acceptable by the study volunteers. A 3-day sampling interval did not allow characterization of acute changes in hormone production during the IOI. In addition, study visits were less frequent when LUFs persisted long after the expected day of the second ovulation of the IOI.
WIDER IMPLICATIONS OF THE FINDINGS
Information about the growth and endocrine dynamics of OvFs and LUFs developing in unstimulated cycles in women may be applied to the early detection of LUF-associated anovulatory infertility and clinical management of women with this condition.
STUDY FUNDING/COMPETING INTEREST(S)
No external funding sources were used for this study. The authors have no conflicts of interest in publishing this manuscript.
TRIAL REGISTRATION NUMBER
ClinicalTrials.gov Identifier: NCT01389141.
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