Background
The primary roles of the ovaries are to support the growth and maturation of oocytes for the acquisition of fertilizability and competence for embryonic and fetal development, as well as the production of sex steroid hormones to induce the estrous cycle and sustain pregnancy. These ovarian functions are regulated by gonadotrophins and steroid hormones. In mono-ovulatory species, the emergence of follicular growth is induced by the surge-like secretion of follicle-stimulating hormone (FSH). A dominant follicle is then selected as the decrease in the level of FSH by the inhibitory effects of estradiol-17β (E
2) and inhibin secreted by the follicles themselves. The dominant follicle continues to grow due to the stimulation by luteinizing hormone (LH), resulting in ovulation [
1,
2]. Most follicles degenerate during follicular growth, and only a small proportion of follicles develop and ovulate [
1,
2].
The ovarian reserve, the pool of primordial follicles in a pair of ovaries in individuals, is defined as the potential ability of ovarian function [
3,
4] and is an indicator of female fertility in mono-ovulating species, such as humans [
4] and cattle [
5]. The peak number of antral follicles in a pair of ovaries during follicular waves counted by ultrasonography (the antral follicle count; AFC) positively correlates with the number of primordial follicles [
6] and may be used to estimate the ovarian reserve [
7]. Although AFC fluctuates during the estrous cycle and markedly varies between individuals, the peak AFC during the estrous cycle shows high repeatability in individual cattle [
7]. Cattle with a high number of antral follicles in a pair of ovaries showed higher reproductive performance, such as higher fertility [
8], a shorter open period [
8], and higher responsiveness to superovulation [
9], than cattle with a low number of antral follicles, even though they were in the same age class. We previously reported that the fertilizability of oocytes after in vitro fertilization (IVF) collected from cattle by ultrasound-guided ovum-pick up (OPU) was higher in high AFC cows having 30 or more antral follicles in a pair of ovaries at the time of OPU than in low AFC cows having less than 30 antral follicles at a 3- or 4-day interval of OPU [
10]. In contrast, when we extended the interval of OPU to 7 days, the fertilizability of oocytes in high AFC cows was impaired and became less than that in low AFC cows, whereas the fertilizability of oocytes derived from low AFC cows was similar regardless of the OPU interval [
10]. These findings indicate that the growth dynamics of antral follicles differ between high and low AFC cows, and the degeneration of antral follicles at the selection phase in the follicular wave may occur earlier in high AFC cows than in low AFC cows. However, the reason for this reversal in the relationship between AFC and oocyte fertilizability remains unclear. Furthermore, we conducted an in vitro growth (IVG) culture of bovine oocyte-cumulus-granulosa complexes (OCGCs) [
11,
12], which enables bovine oocytes without maturational competence from early antral follicles to grow to the stage acquiring competence for maturation and development to the blastocyst stage [
13‐
15] and offspring [
13,
14]. By using this technology, we investigated follicular function, the acquisition of oocyte competence, and steroidogenesis in granulosa cells, and estimated follicular growth dynamics from the period during which follicles cannot be detected by ultrasonography in vivo to the period during which oocytes acquire developmental competence in high and low AFC cows. Consequently, OCGCs derived from early antral follicles (0.5–1.0 mm in diameter) in the high AFC group having 25 or more antral follicles (≥2.0 mm in diameter) in an ovary collected at a slaughterhouse showed higher oocyte maturational competence and fertilizability than those in the low AFC group having less than 25 antral follicles [
11,
12]. Although the proliferation of granulosa cells was the same in both groups, E
2 production by OCGCs was higher in the high AFC group than in the low AFC group [
12]. We also revealed that granulosa cells surrounding in vitro-grown oocytes having higher maturational competence secreted more E
2 and less progesterone (P
4) than those surrounding less competent in vitro-grown oocytes using medium containing androstenedione (A
4) instead of E
2 [
16].
Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-β family. AMH is known to be a marker of ovarian reserve, and there is a strong correlation between AFC and AMH in human [
17,
18] and cattle [
19,
20]. AMH is secreted by the granulosa cells of primary to early antral follicles [
17]. Some researchers conducted comparative studies of predictive values for human ART between AFC and blood AMH level, and suggested that predictive values of AFC and AMH were similar [
4,
21‐
27]. In addition, some studies indicated that the predictive value of AFC was higher than that of AMH [
28‐
30], although other studies demonstrated the contradictory results [
18,
31,
32]. In AMH-deficient mice, the premature depletion of primordial follicles occurred [
33], and AMH inhibited the activation of primordial follicles in cattle [
34]. AMH inhibited the FSH-stimulated growth of antral follicles and E
2 production by decreasing the sensitivity of preantral and antral follicles to FSH in mice [
35], humans [
36,
37], and sheep [
38]. These findings suggest that AMH is an important regulator of follicular activation, follicular growth, and steroidogenesis in growing follicles. Furthermore, the plasma concentration of AMH positively correlated with the number of primordial follicles and AFC in cattle [
6] and humans [
17]. In cattle, the concentration of AMH in the follicular fluid of antral follicles (≥3 mm in diameter) decreased during follicular growth [
39,
40]. Granulosa cells derived from antral follicles (3–5 mm in diameter) produced more E
2 and AMH in high AFC cows having 25 or more follicles in a pair of ovaries than in low AFC cows having 15 or fewer antral follicles regardless of the addition of FSH to the in vitro culture of granulosa cells [
41]. In the follicular fluid of antral follicles (5–7 mm in diameter), immediately before the selection of dominant follicles, AMH concentrations were similar between high AFC heifers and low AFC heifers, while E
2 concentrations were lower in high AFC heifers than in low AFC heifers [
42]. On the other hand, E
2 concentrations in the follicular fluid of ovulatory follicles (approximately 15 mm in diameter) were higher in high AFC heifers than in low AFC heifers [
43]. These findings indicated that AMH regulates FSH-stimulated E
2 production during follicular growth, and this regulation may differ between each follicular growth stage. However, there is currently no information on the relationship between AMH concentrations in follicles after selection (≥8 mm in diameter) or before recruitment (< 4 mm in diameter) and AFC. In the present study, we investigated the relationship between AFC, follicular growth dynamics, FSH concentrations in plasma and steroid hormones, and E
2, testosterone (T, one of the precursors of E
2), and P
4 concentrations in plasma and follicular fluid as the factors affecting oocyte developmental competence in high and low AFC cattle. We also investigated the relationship between AMH and AFC at follicular stages before recruitment by the IVG of OCGCs derived from early-antral follicles (< 1 mm in diameter) and ultrasound-guided follicular aspiration, respectively.
Discussion
In our previous study [
10], the normal fertilizability of oocytes was higher in the high AFC group than in the low AFC group in the 3- or 4-day interval of OPU-IVF, while this result was reversed in the 7-day interval of OPU-IVF wherein the normal fertilizability of oocytes was higher in the low AFC group than in the high AFC group. In the present study, the number of intermediate follicles increased after follicular ablation and then decreased within a few days in the high AFC group; approximately 3 to 4 days after follicular ablation, the number of intermediate follicles peaked in the high AFC group (Fig.
2a). This result indicates that most follicles 3–4 days after follicular ablation were in the growing phase in the high AFC group, resulting in the higher fertilizability of oocytes, as described in our previous study [
10]. However, 7 days after follicular ablation, follicles already start to regress and oocyte fertilizability becomes low. In the low AFC group, the number of intermediate follicles was stable regardless of the number of days after follicular ablation. In the present study and a previous study [
7], FSH concentrations were higher in low AFC cows than in high AFC cows. These results indicate that intermediate follicles in the low AFC group are consistently growing in the presence of a high FSH concentration, resulting in higher fertilizability in the low AFC group than in the high AFC group at the 7-day interval of OPU. The early degradation of intermediate antral follicles may be caused by higher E
2 concentrations in the dominant follicle in the high AFC group, which may induce the degeneration of subordinate follicles [
54].
In the present study, E
2 concentrations and the E
2/P
4 ratio in follicular fluid at the ovulatory phase were higher in the high AFC group (1127 ng/mL) than in the low AFC group (332 ng/mL). Mossa et al. [
43] also reported higher E
2 concentrations in the dominant follicle in high AFC heifers (588 ng/m) than in low AFC heifers (435 ng/mL). A previous study using an in vitro culture of granulosa cells suggested that the lower expression levels of FSH receptors and aromatase (P450arom) resulted in impaired responses to FSH and E
2 production by granulosa cells in low AFC cattle [
41]. These findings suggest a difference in responses to a FSH stimulus between high and low AFC cattle. However, Ireland et al. [
42] demonstrated that E
2 concentrations in follicles (5–7 mm) at the emergence of the follicular wave (24 to 48 h after ovulation) were higher in low AFC heifers (approximately 90 ng/mL) than in high AFC heifers (approximately 40 ng/mL). In the present study, E
2 concentrations in follicles of > 8 mm in diameter at the selection phase were 168 ng/mL in low AFC cows and 203 ng/mL in high AFC cows. These results indicate that the function of granulosa cells in follicles in low and high AFC cattle is altered before and after the expression of LH receptors at approximately 8 mm [
49]. Furthermore, these results suggest that the ability of LH-mediated E
2 production is impaired in the low AFC group, resulting in lower E
2 concentrations in dominant follicles after the selection phase. Endo et al. [
55] reported that E
2 promoted the growth and maturational competence of bovine IVG oocytes. Our previous findings indicated that E
2 production was higher by OCGCs producing matured oocytes after in vitro maturation (IVM) than by OCGCs producing immature oocytes after IVM [
16]. Moreover, OCGCs derived from high AFC ovaries showed higher E
2 production by granulosa cells and higher oocyte developmental competence than those from low AFC ovaries [
12]. Consequently, impaired E
2 production in low AFC cattle may have a negative impact on the growth, maturation, and developmental competence of oocytes, resulting in lower fertility in low AFC cattle than in high AFC cattle.
E
2 and T concentrations were higher in the high AFC group than in the low AFC group not only in follicular fluid, but also in plasma, whereas FSH concentrations were higher in the low AFC group than in the high AFC group in the present study. Previous studies reported that T plasma concentrations were higher in the high AFC group than in the low AFC group in heifers and cows [
43] and FSH plasma concentrations were higher in the low AFC group than in the high AFC group in heifers [
9] and cows [
7,
56]; however, E
2 plasma concentrations were similar in low and high AFC cattle [
7,
9,
56]. A possible reason for the difference in E
2 plasma concentrations between the present and previous studies is the difference in the age of cattle used in experiments. In the present study, we used older cows (3.7, 11.4, and 14.5 years old in low AFC cows; 3.9, 4.8, 11.8, and 12.9 years old in high AFC cows) than those in previous studies (14–33 months old [
9], 3–5 years old [
7], and 2.6–10.8 years old [
56]). In cattle, the numbers of primordial and preantral follicles are stable after birth until 4 to 6 years old and then decrease [
57]. In humans, E
2 serum concentrations begin to decrease and FSH serum concentrations markedly increase 2 years before the last menstrual period [
58]. In the present study, average E
2 plasma concentrations from days 0 to 16 were similar in low AFC cows (3.2 ± 1.5 pg/mL) and high AFC cows (3.8 ± 1.9 pg/mL) younger than 10 years old; however, they were higher in high AFC cows (4.3 ± 1.8 ng/mL) than in low AFC cows (3.0 ± 1.4 ng/mL) older than 10 years (
P < 0.01, the Student’s
t-test). These results indicate an age-related decrease in E
2 plasma concentrations, particularly in low AFC cows, and that the fertility of cows decreases at younger ages in low AFC cows than in high AFC cows. We speculate that if we use only young age cows, there will be no difference in E
2 plasma concentrations between the groups, and if we use only old age cows, E
2 plasma concentrations will be higher in the high AFC cows. In addition to E
2, inhibin is a major hormone causing negative feedback on FSH secretion [
59]. A previous study using 3–5-year-old cows [
7] suggested that inhibin-A serum concentrations were slightly higher in high AFC cows than in low AFC cows at the ovulatory phase (
P = 0.07), but not at the selection phase of dominant follicles. Another study using 11–13-month-old heifers [
42] indicated that inhibin-A concentrations in follicles (5–7 mm) at the emergence of the follicular wave (24 to 48 h after ovulation) were similar between high and low AFC heifers. Future studies are needed to investigate the relationship between AFC, age, and the competence of E
2 and inhibin production in granulosa cells.
In the present study, AMH concentrations in follicular fluids derived from large follicles (≥8 mm) at different stages of follicular growth (selection, luteal, and ovulatory phases) were slightly higher in the high AFC group than in the low AFC group. Furthermore, AMH concentrations in the IVG media of OCGCs derived from a 4- to 12-day culture were higher in the high AFC group than in the low AFC group. Scheetz et al. [
41] reported that the production of AMH and expression of the messenger ribonucleic acid of AMH were greater in cultured granulosa cells derived from high AFC cows than those from low AFC cows. These findings indicate that the ability to produce AMH by granulosa cells is higher in high AFC cows than in low AFC cows throughout follicular development. On the other hand, AMH decreased the expression of FSH receptors in human granulosa cells [
60], and E
2 production was impaired by decreasing the responses of preantral and antral follicles to FSH in mice [
35], humans [
36,
37], and sheep [
38]. In the present study and a previous study [
43], higher E
2 concentrations in follicular fluid were observed in the high AFC group, while the AMH concentration that suppressed E
2 secretion was higher in the high AFC group than in the low AFC group. The reason for the contradiction of AMH and E
2 concentrations may be explained by T concentrations in follicular fluid. T has been shown to increase the transcription of FSH receptors in bovine cultured granulosa cells [
61], and the in vivo results of the present study showed higher T concentrations in the high AFC group. These results suggest that higher T production by theca cells counteracts the function of AMH for reducing FSH-mediated E
2 production in high AFC cattle. The roles of theca cells in follicular growth need to be investigated in more detail.
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