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The aim of the present study was to characterize the effect of long-term usage of dienogest, a fourth-generation progestin that possesses progestogen and anti-androgen activities, on the stockpile of oocytes and fertility after administration. Female ICR mice (100 days old) were divided into a dienogest group and a control group. The mice received 16 consecutive subcutaneous injections of 5 mg dienogest dissolved in corn oil or corn oil as a vehicle control every 4 days. The mice treated with dienogest had more total offspring and larger litter sizes after the final administration than the mice treated with the vehicle control. Greater numbers of primordial follicles were detected at both 4 and 80 days after the final administration. No significant differences were found in serum anti-Müllerian hormone concentrations at 4 and 80 days after the final dienogest administration. The ratio of primary to primordial follicles was decreased in 3-day-old newborn ovaries cultured for 4 days with dienogest (10–7, 10–6 and 10–5 mol/l) compared with ovaries cultured without dienogest. The results of the present study indicate that dienogest suppresses the activation of primordial follicles during its administration and preserves the primordial follicle stockpile and subsequent fertility in mice.
). Thus, the development of strategies to prevent age-related oocyte depletion may preserve or improve the fertility of women attempting pregnancy in their late 30s and early 40s.
Oocytes are preserved as dormant primordial follicles, which are a complex consisting of an immature oocyte arrested at the diplotene stage of meiosis and several surrounding flattened somatic cells, in the ovary (
). The activation of primordial follicles is the initial step in the complex process of oocyte growth, and is accompanied by the proliferation and differentiation of the surrounding pre-granulosa cells (
). The molecular mechanism underlying the activation of primordial follicles has been previously described. The importance of the phosphatase and tensin homologue (PTEN)/phosphatidylinositol-3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDPK1)/v-akt murine thymoma viral oncogene homologue 1 (AKT1) system, which is related to KIT ligand, and bone morphogenetic protein (BMP) and anti-Müllerian hormone (AMH)/SMAD signalling has been demonstrated (
Testosterone induces redistribution of forkhead box-3a and down-regulation of growth and differentiation factor 9 messenger ribonucleic acid expression at early stage of mouse folliculogenesis.
Dienogest (DNG) is a fourth-generation progestin that possesses progestogen and anti-androgen activities, whereas it lacks oestrogenic, glucocorticoid and anti-mineral corticoid effects (
Dienogest is a selective progesterone receptor agonist in transactivation analysis with potent oral endometrial activity due to its efficient pharmacokinetic profile.
), it can improve androgenic symptoms such as acne and hirsutism. DNG is used as a contraceptive and for the treatment of endometriosis in women of reproductive age (
). Because of its anti-androgen action, DNG is considered favourable for women and the usage of DNG has been increasing worldwide. As DNG is used for several or more years in a portion of patients, its cumulative effects on organs, if any, may be of concern.
In women, DNG has been reported to show inhibitory effects on follicular growth without affecting the pulsatile secretion of LH or basal levels of LH and FSH (
). Moreover, a single oral administration of DNG induced atresia of dominant follicles with apoptotic changes and the loss of aromatase expression in granulosa cells of cynomolgus monkeys (
Dienogest, a selective progestin, reduces plasma oestradiol level through induction of apoptosis of granulosa cells in the ovarian dominant follicle without follicle-stimulating hormone suppression in monkeys.
). The modification of follicle growth and subsequent sex steroid hormone production is thought to affect the profile of a number of cytokines including Kit ligand and BMP, which are well-known activators of primordial follicles in the ovary (
). Furthermore, the anti-androgen action of DNG may negatively affect fertility, as androgen action has been reported to modulate primordial follicle activation (
Testosterone induces redistribution of forkhead box-3a and down-regulation of growth and differentiation factor 9 messenger ribonucleic acid expression at early stage of mouse folliculogenesis.
). As stated, DNG potentially possesses multiple effects on the activation of primordial follicles. However, the effects of long-term usage of DNG on the oocyte stockpile and fertility following administration have not yet been clarified. Thus, the effects and safety of DNG with respect to fertility require further elucidation. The aim of the present study is to elucidate the effect of long-term usage of DNG on the stockpile of oocytes and fertility after administration.
Materials and methods
Ethics and animal care
All procedures were carried out in accordance with the animal care and welfare guidelines of the Shiga University of Medical Science. All experimental procedures were approved by the Shiga University of Medical Science Animal Experimentation Centre Committee on 6 November 2015 (reference number:150–89). All mice (ICR mice or C57/B6 mice) were purchased from CLEA Japan, Inc. (Tokyo, Japan), and were maintained on a 12-h light, 12-h dark cycle and provided food and water ad libitum.
DNG dosing rationale for adult mice
In order to confirm the effect of DNG on ovarian function, 100-day-old ICR mice were given three consecutive subcutaneous injections of 5 mg DNG (Mochida Pharmaceutical Co., Japan) dissolved in 0.2 ml corn oil, with 4 days between injections. After the final injection (48 or 96 h), the mice were killed by an anaesthesia overdose of ketamine and xylazine to obtain the ovaries. The ovaries were paraffin embedded, sectioned at 4 µm and stained with haematoxylin and eosin (H&E). Ovarian morphology was assessed to determine whether DNG suppresses the growth of antral follicles.
Experimental schema for adult mice
The schema of the three experiments for adult mice following establishment of the DNG dosage rationale is shown in Figure 1.
Figure 1Schematic representation of the experimental protocols for adult mice. The schema of the three experiments for adult mice following establishment of the DNG dosage rationale is shown.
Mice that were 100 days old were treated with subcutaneous injection of 5 mg of DNG (dissolved in 0.2 ml corn oil; DNG group) or corn oil (control group) every 4 days for a total of 16 administrations until the mice were 160 days old. Starting at 164 days of age, each female mouse was housed with a male mouse of the same age, with 11 pairs initially established for each group. The mice were observed each morning, and litter sizes and parturition frequency were recorded. The mice were observed for at least 60 days following the last delivery, and the last delivery age of the mouse was determined when the mouse did not give birth for 60 days.
Follicle categorization and enumeration, and determination of AMH level
The effects of DNG on the primordial follicle stockpile and follicular development were examined. Forty 100-day-old mice were treated with 16 doses of DNG (as above) and the mice were killed 4 or 80 days after the last DNG injection. After killing the animals, the ovaries were collected, and blood serum was sampled.
The investigator was blinded to the treatment groups until all procedures were completed and all results were obtained. Ovaries were fixed in Bouin solution for 5 min, embedded in paraffin, sectioned at 4 µm and stained with H&E. Procedures for follicle identification and counting were conducted as previously reported by
, with the following modifications. Briefly, oocytes/follicles were counted in every 10th section throughout the entire ovary when the nucleus was present in the section. In the present study, primordial follicles were defined as the combination of two categories previously reported: resting primordial follicles surrounded by only flattened pre-granulosa cells, and primordial to primary transition follicles that have just initiated development and contain some cuboidal granulosa cells. A correction factor was used to determine total oocyte/follicle numbers for primordial follicles to primary follicles because the oocyte nucleus of primordial and primary follicles was about 6–8 µm and oocytes were counted on every sixth 4 µm section; thus, the numbers of enumerated primordial and primary follicles were multiplied by 10. Because the nuclear diameter of oocytes in secondary to antral follicles was typically greater than 20 µm, the number of enumerated secondary and antral follicles was multiplied by two.
The blood samples were centrifuged at 20,000g for 10 min at 4°C, and serum was obtained and frozen until use. The serum concentration of AMH was measured using an ELISA kit (ELISA Kit for Anti-Müllerian Hormone; Cloud-Clone Corp., Houston, TX, USA), according to the manufacturer's instructions.
Neonatal ovary culture and primordial/primary follicle enumeration
Untreated pairs of female and male C57/BL6 mice were housed and monitored daily. When a female mouse had a large number of female newborns in a parturition, the following culture experiments using the ovaries of the newborns were performed. We defined day 0 as the day each mouse was born. Postnatal day 3 female mice were killed and their bilateral ovaries were harvested. Each ovary was initially washed in DMEM/Ham's F12 medium (Thermo Fisher Scientific, MA, USA) supplemented with 100 mIU/ml penicillin and 50 µg/ml streptomycin (Wako Pure Chemical Industries, Japan). All the ovaries from the litter of one mother were pooled in a dish and then randomly divided into groups for treatment with either DNG (0, 10–8, 10–7, 10–6 or 10–5 mol/l), norethindrone (NET) (Sigma-Aldrich, MO, USA), a first-generation progestin possessing androgen activity (0, 10–6 or 10–5 mol/l), or flutamide (Sigma-Aldrich), a synthetic non-steroidal anti-androgen (0, 100 or 1000 ng/ml). The experiments using NET and flutamide were designed to examine the specificity of DNG on the primordial follicle activation in vitro, the concentrations of flutamide (0, 100 or 1000 ng/ml) being determined based on effective blood concentrations in humans (
: https://www.drugs.com/pro/flutamide.html). After assignment to the groups, whole ovarian tissues were individually cultured on floating filters (0.4 µm Millicell-CM, Merck Millipore, MA, USA) in 3.5 ml Dulbecco's modified Eagle's medium/Ham's F-12 medium (1:1 v/v) containing 0.1% BSA (Sigma-Aldrich), 1% ITS (550 ng/ml transferrin, 1 µg/ml insulin, 670 pg/ml sodium selenite) (Thermo Fisher Scientific), 100 mIU/ml penicillin and 50 µg/ml streptomycin (Wako Pure Chemical Industries) with the indicated concentrations of DNG, NET or flutamide, in six-well culture plates (Thermo Fisher Scientific). The neonatal ovary cultures were maintained at 37°C in a humidified incubator under 5% CO2 in air for 4 days, with the culture medium changed every second day. Neonatal ovaries cultured for 4 days were used for the morphological assessment.
Ovaries were fixed in Bouin solution for 2 min and processed as for adult mouse tissues. Although the samples were collected every five sections, only six sections from each ovary (the 2nd, 3rd and 4th from the beginning of the section, and the 4th, 3rd and 2nd from the end of the section) were used to count follicles, as a portion of the follicles in the centre of the ovary underwent atresia. The proportion of primary follicles (defined when the long diameter of the oocyte was 25 µm or more and the oocyte was surrounded by single layer of granulosa cells) per primordial follicle (defined when the long diameter of the oocyte was less than 25 µm) was calculated for each ovary.
Phosphorylation of AKT in cultured neonatal ovaries with exposure to DNG
Postnatal day 3 female mice were killed, and their bilateral ovaries were harvested and cultured for 4 days after random allocation to treatment with three different DNG concentrations (0, 10–6 or 10–5 mol/l), as described for the neonatal ovary culture and primary/primordial follicle enumeration experiments. After 4 days of culture, the ovaries were frozen and kept at −80°C until use, and this procedure was repeated until a sufficient number of ovaries had been pooled. Protein was extracted from four ovaries exposed to the same concentration of DNG, in 30 µl of RIPA Lysis Buffer using a sonicator. Fifteen micrograms of protein (in an average of 15–25 µl) were boiled in Laemmli solution (Wako Pure Chemical Industries) under reducing conditions, at 95°C for 5 min. Samples were separated on a 4–20% gradient SDS–polyacrylamide gel (Wako Pure Chemical Industries), and then transferred to a polyvinylidene difluoride membrane (Merck Millipore). Membranes were incubated with Blocking One Solution (Nacalai Tesque, Japan) at room temperature for 1 h to block non-specific binding, followed by incubation with rabbit anti-mouse phospho-AKT (Cat # 9271; 1:1000, Cell Signaling Technology, MA, USA) diluted in Can Get Signal Solution 1 (Toyobo, Japan) at 4°C overnight. After washing with TBST for 20 min three times, membranes were exposed to peroxidase-labelled anti-rabbit IgG (1:1000; GE Healthcare, IL, USA) in Can Get Signal Solution 2 (Toyobo) for 1 h at room temperature. Peroxidase labelling was detected by chemiluminescence using the Chemi-Lumi One Super (Nacalai Tesque). After exposure to stripping buffer, membranes were subsequently incubated with rabbit anti-mouse AKT (Cat # 9272; 1:1000, Cell Signaling Technology) followed by the same procedure as described above for the detection of phospho-AKT. Image J (National Institutes of Health, USA) was used to quantify the ratios of phospho-AKT to AKT.
Statistical analysis
All data points represent the mean of at least three independent measurements and are expressed as the mean ± standard error of the mean (SEM). GraphPad Prism version 7 (GraphPad Software, CA, USA) was used to perform a Student's t-test, with a significance threshold of P < 0.05.
Results
Establishment of DNG dosing rationale
Ovarian morphology was determined following treatment (three doses at 4-day intervals) with DNG or corn oil. Large antral follicles were not observed in the ovaries 48 or 96 h after the final DNG administration, and only medium-sized antral follicles with a number of pyknocytotic granulosa cells were observed. Furthermore, there was no particular difference observed in the morphological features of secondary, primary and primordial follicles (Supplementary Figure S1A–D). Thus, DNG suppressed ovulation and caused medium-sized antral follicles to decline into atresia. The administration of 5 mg DNG every 4 days was used in subsequent experiments.
Fertility following DNG administration
The total number of lifetime offspring following the 16 administrations of DNG was significantly greater than the control group (Figure 2A, 60.4 ± 7.4 in Control versus 83.9 ± 5.8 in DNG, P = 0.02), whereas the age of last parturition (duration of fecundity in a lifetime) and number of parturitions over a lifetime did not differ between the groups (Figure 2B and C, respectively). Only the average litter size (number of pups per parturition) was larger in the DNG group (Figure 2D, 9.10 ± 0.83 in Control versus 12.14 ± 0.48 in DNG, P = 0.0048), which accounted for the increased cumulative number of newborns.
Figure 2Effect of DNG administration on fertility in mice. A Total lifetime offspring of mice treated with DNG or corn oil as a vehicle control (Cont). B The age of last parturition of mice treated with DNG or Cont. C The number of parturitions in the lifetime of mice treated with DNG or Cont. D The average litter size in mice treated with DNG or Cont. Although 11 pairs of mice were initially used for each group, only 7 control and 9 DNG pairs were available at the end of the study due to unexpected mortality. Data are presented as mean ± SEM, with P < 0.05 considered to be statistically significant; N indicates sample size (the number of mice per group).
Follicle categorization and enumeration, and serum level of AMH
Ovarian morphology is shown in Figure 3A–D. No apparent differences in ovarian morphology were observed among the groups. In 164-day-old mice, the numbers of primordial and antral follicles in the DNG group were greater than in the control group, although the numbers of primary and secondary follicles were no different (Figure 3E). The number of primordial follicles was greater in the 240-day-old mice of the DNG group, although the numbers of primary, secondary and antral follicles were no different (Figure 3F). The serum AMH levels did not differ between the groups in 164-day-old-mice and 240-day-old mice (Figure 4A and B).
Figure 3Ovarian morphology and the number of classified follicles in 164-day-old and 240-day-old mice (4 and 80 days after final administration, respectively). The corn oil-treated group and the DNG-treated group are indicated as ‘Control’ and ‘DNG’, respectively. Primordial follicle, primary follicle, secondary follicle and antral follicle are indicated as ‘Prmo’, ‘Pri’, ‘Sec’ and ‘Ant’, respectively. A, C Ovarian morphology in 164-day-old and 240-day-old corn oil-treated mice. B, D Ovarian morphology in 164-day-old and 240-day-old DNG-treated mice. Scale bar = 200 µm. E, F Classification and enumeration of follicles in 164-day-old and 240-day-old mice, respectively. Four mice in each group were used. Data are presented as mean ± SEM, with P < 0.05 considered to be significant; N.S., not significant.
Figure 4Serum AMH levels in mice at 164 and 240 days old. A Serum AMH levels at 164 days old in DNG- and corn oil-treated mice. B Serum AMH levels at 240 days old in DNG- and corn oil-treated mice (Cont). Data are presented as mean ± SEM, with P < 0.05 considered to be significant. N.S., not significant; N indicates sample size (the number of mice per group).
In the in-vitro culture of mouse ovaries, a number of primary follicles appeared as large round cells in the centre of the ovarian sections from the control group, whereas these primary follicles appeared to be reduced or were obviously reduced in the DNG group (Supplementary Figures S2A–J). The ratio of primary to primordial follicles was significantly lower at DNG concentrations of 10–5 to 10–7 mol/l in the culture media compared with control, whereas this difference disappeared at 10–8 mol/l DNG (Figure 5A). No significant difference in the primary to primordial follicle ratio was seen in response to NET treatment (10–5 to 10–6 mol/l) (Figure 5B, Supplementary Figures S3A–F), whereas it was significantly lower in ovaries incubated with flutamide (100–1000 ng/ml) (Figure 5C, Supplementary Figures S3G–L).
Figure 5The ratio of primary to primordial follicles in the presence of DNG, NET or flutamide. A The proportion of primary to primordial follicles in the presence of different concentrations of DNG (0, 10–8, 10–7, 10–6 and 10–5 mol/l). B The proportion of primary to primordial follicles in the presence of various NET concentrations (0, 10–6 and 10–5 mol/l). C The proportion of primary to primordial follicles in the presence of various flutamide concentrations (0, 100 and 1000 ng/ml). Data are presented as mean ± SEM, with P < 0.05 considered to be statistically significant; N.S., not significant; N indicates sample size (the number of ovaries per group).
Effect of DNG on phosphorylation of AKT in cultured neonatal ovaries
The phosphorylation of AKT was significantly decreased at DNG concentrations of 10–5 to 10–6 mol/l in the culture media compared with controls (Supplementary Figure S4).
Discussion
In the present study, the effect of DNG on fertility compared with the vehicle control was confirmed using different methods. First, the total number of newborns and the litter sizes increased following DNG administration. Second, a greater number of primordial follicles were maintained at 4 and 80 days after DNG administration. Third, the ratio of primary to primordial follicles was reduced by DNG in vitro. These results suggest that DNG does not adversely affect reproduction, but in fact suppresses the activation of primordial follicles, thereby preserving the primordial follicle stockpile and future fertility.
The control of primordial follicle activation could provide two contrasting strategies for fertility treatment. The activation of primordial follicles increases the growing follicle pool, resulting in increased availability of oocytes for the treatment of infertility in patients with premature ovarian insufficiency or who are poor responders to stimulation medications. This strategy is based on the theory that a large proportion of the few remaining primordial follicles are forced into being activated, thereby obtaining more oocytes prior to IVF oocyte retrieval.
On the other hand, the suppression of activated primordial follicles is a potential strategy for preserving the oocyte stockpile in women who wish to delay pregnancy. The present strategy is based on the theory that daily expenditure of primordial follicles is reduced, and more primordial follicles are preserved for the future by suppressing primordial follicle activation. The present study showed that DNG represents a candidate agent that fulfils the aforementioned criteria.
Unfortunately, the serum levels of DNG in mice could not be measured in the present study. Thus, establishment of the dosage rationale was based on ovarian responses to DNG. The clinical dose of DNG for contraception and treatment of endometriosis is 2 mg/day. Ovulation is inhibited at this dosage and only small antral follicles are observed in the ovary. In a preliminary experiment of the present study, administration of 1 mg DNG every 4 days to mice allowed some follicles to develop into large antral follicles (data not shown), whereas administration of 5 mg DNG every 4 days completely inhibited follicular growth to medium-sized antral follicles, as shown in SupplementaryFigure 1. In contrast to primates and humans, a much higher dose of progestin is needed to regulate follicular growth and ovulation in rodents. The necessity for high doses of progestin is thought to be due to the absence of an enterohepatic circulation system, which results in progestin not being reabsorbed from the intestine and quickly metabolized, as well as differences in progestin potency in rodents. Intramuscular administration of 100 mg of DNG/kg/day to rats was reported to reduce ovarian weight, whereas 10 mg of DNG/kg/day did not (
). For these reasons, we employed 5 mg of DNG every 4 days to mice to produce a similar status in the ovary to that of the human ovary exposed to 2 mg of DNG daily.
In the present study, primordial follicle activation was suppressed in mice under conditions where antral follicle growth and ovulation were suppressed by DNG administration in vivo. Furthermore, in ovaries cultured with a wide range of DNG concentrations, from 10–7 mol/l (equivalent to concentrations achieved in humans in vivo) to 10–5 mol/l, the ratio of primary to primordial follicles was decreased. Taken together, the activation of primordial follicles appeared to be suppressed in mice by specific concentrations of DNG, which mirrored the observed serum concentrations in human patients treated with DNG. However, the clinical significance of DNG in suppressing primordial follicle activation needs to be clarified in humans using appropriate experimental methodologies in the future.
At 4 days after the end of DNG administration, the level of AMH was similar between the DNG and control groups, whereas the number of antral follicles was significantly greater, and the absolute number of secondary follicles was greater in the DNG group. AMH is a marker of the remaining follicle pool, because serum AMH levels are positively correlated with the size of the remaining primordial follicle pool, although AMH is actually produced by late secondary to early antral phase follicles (
). Thus, serum AMH levels should be higher in the DNG group 4 days after the end of DNG administration. Progestin administration has been reported to decrease serum AMH levels in vivo (
Dienogest, a selective progestin, reduces plasma oestradiol level through induction of apoptosis of granulosa cells in the ovarian dominant follicle without follicle-stimulating hormone suppression in monkeys.
). One explanation for the lack of observed differences in serum AMH levels immediately following final DNG administration was the decreased ability of granulosa cells to secrete AMH following exposure to DNG.
previously reported that DNG prevents consumption of the primordial stockpile attributable to the adverse effect of cyclophosphamide. Cyclophosphamide was recently reported to reduce primordial follicle numbers by ‘burn out’', activating large numbers of primordial follicles that immediately fall into atresia (
). The mechanism of the protective effect of DNG against cyclophosphamide may underlie its suppressive effect on primordial follicle activation observed in the present study as well.
We showed that DNG did not detrimentally affect fertility in mice, but rather, it preserved the stockpile of primordial follicles by suppressing primordial follicle activation. However, the mechanistic basis for this effect on the ovary remains unknown. Because androgens are known to promote primordial follicle activation, we also examined the effect of NET, a first-generation progestin possessing androgen action, and flutamide, a non-steroidal anti-androgen, on primordial follicle activation. The results demonstrated that the suppressive effect on primordial follicle activation was seen only with DNG and flutamide, and not with NET. There are reports that neonatal ovary expresses cytochrome P450 17 alpha-hydroxylase (P450c17), a key enzyme for the production of androgen, and indeed, that neonatal ovaries can produce testosterone. There are also studies showing that androgen receptors are expressed in the neonatal ovary and that the exposure of neonatal ovaries to androgens affects ovarian biology and future ovarian function (
Testosterone induces redistribution of forkhead box-3a and down-regulation of growth and differentiation factor 9 messenger ribonucleic acid expression at early stage of mouse folliculogenesis.
). Based on such observations, one explanation for the suppressive effect of DNG on primordial follicle activation may lie in its anti-androgen actions.
Before the experiment, we expected the activation of primordial follicles to primary follicles in the neonatal ovaries cultured with NET, because testosterone has been reported to promote primordial follicle activation in vitro (
Testosterone induces redistribution of forkhead box-3a and down-regulation of growth and differentiation factor 9 messenger ribonucleic acid expression at early stage of mouse folliculogenesis.
Dienogest is a selective progesterone receptor agonist in transactivation analysis with potent oral endometrial activity due to its efficient pharmacokinetic profile.
Dienogest, a selective progestin, reduces plasma oestradiol level through induction of apoptosis of granulosa cells in the ovarian dominant follicle without follicle-stimulating hormone suppression in monkeys.
) as we stated. However, our findings showed that NET did not in fact promote activation in vitro. Although there has been no report directly showing differences in androgen activity between NET and testosterone, we know that the androgen action of NET is weaker than testosterone by comparison with the effects of dihydrotestosterone (DHT). DHT possesses approximately 2.4 times the androgen activity of testosterone (
Dienogest is a selective progesterone receptor agonist in transactivation analysis with potent oral endometrial activity due to its efficient pharmacokinetic profile.
Dienogest, a selective progestin, reduces plasma oestradiol level through induction of apoptosis of granulosa cells in the ovarian dominant follicle without follicle-stimulating hormone suppression in monkeys.
). Thus, testosterone can be concluded to be approximately 20 times stronger than NET with respect to androgen action. A greater number of primordial follicles were reported to be activated in vitro when mouse neonatal ovary was cultured with 10–5M of testosterone for 10 days. In the present study, neonatal ovary was cultured at 10–5 or 10–6 M of NET for 4 days. One explanation for the difference in results between the previous study using testosterone and the present study is the difference in effective androgen activity and period of culture.
In this study, we confirmed that the phosphorylation of AKT, which is representative of PI3K signalling, is suppressed in neonatal ovary by exposure to DNG. PI3K signalling is thought to be the main pathway regulating primordial activation, and its activation by DNG may either be direct or indirect. Further research, especially using in-vitro experiments, is required to increase our understanding of the potential role of anti-androgen action in regulating ovary reserves.
Taken together, the results of the present study indicate that DNG suppresses the activation of primordial follicles, and preserves the primordial stockpile and subsequent fertility in mice.
Acknowledgement
This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (grant number C-23592400).
Appendix. Supplementary material
The following is the supplementary data to this article:
Ovarian morphology 48 and 96 h after DNG administration. Ovaries 48 h after DNG administration, observed with A low and B high magnification; and 96 h after DNG administration, observed with C low and D high magnification. Scale bar = 200 µm.
Morphology of neonatal mouse ovaries cultured with or without DNG. A, B Morphology of neonatal ovaries cultured in the absence of DNG, observed with low and high magnification, respectively. C, D Morphology of neonatal ovaries cultured in the presence of 10–8 mol/l DNG, observed with low and high magnification, respectively. E, F Morphology of neonatal ovaries cultured in the presence of 10–7 mol/l DNG, observed with low and high magnification, respectively. G, H Morphology of neonatal ovaries cultured in the presence of 10–6 mol/l DNG observed with low and high magnification, respectively. I, J Morphology of neonatal ovaries cultured in the presence of 10–5 mol/l DNG observed with low and high magnification, respectively. The number of primary follicles appearing as large round cells in the centre of the ovarian sections (indicated by arrows in panels B and D) appeared to be greater in the absence and 10–8 mol/l of DNG.
Morphology of neonatal mouse ovaries cultured with NET or flutamide. A, B Morphology of neonatal ovaries cultured in the absence of NET, observed with low and high magnification, respectively. C, D) Morphology of neonatal ovaries cultured in the presence of 10–6 mol/l NET, observed with low or high magnification, respectively. E, F Morphology of neonatal ovaries cultured in the presence of 10–5 mol/l NET observed with low and high magnification, respectively. G, H Morphology of neonatal ovaries cultured in the absence of flutamide, observed with low and high magnification, respectively. I, J Morphology of neonatal ovaries cultured in the presence of 100 ng/ml flutamide observed with low and high magnification, respectively. K, L Morphology of neonatal ovaries cultured in the presence of 1000 ng/ml flutamide, observed with low or high magnification, respectively. Primary follicles appearing as large round cells in the centre of the ovarian sections are indicated by arrows in panels B, D, F, H, J and L.
Phosphorylation of AKT in cultured neonatal ovary with exposure to DNG. A Western blots of AKT and pAKT in neonatal ovary cultured with 0, 10–6 or 10–5 mol/l DNG. B Quantification of the phosphorylation of AKT. Data are presented as mean ± SEM, with P < 0.05 considered to be significant; N.S., not significant; N indicates sample size.
References
Anesetti G.
Chavez-Genaro R.
Ovarian follicular dynamics after aromatizable or non aromatizable neonatal androgenization.
Dienogest is a selective progesterone receptor agonist in transactivation analysis with potent oral endometrial activity due to its efficient pharmacokinetic profile.
Dienogest, a selective progestin, reduces plasma oestradiol level through induction of apoptosis of granulosa cells in the ovarian dominant follicle without follicle-stimulating hormone suppression in monkeys.
Testosterone induces redistribution of forkhead box-3a and down-regulation of growth and differentiation factor 9 messenger ribonucleic acid expression at early stage of mouse folliculogenesis.
Luyi Zheng completed her postgraduate degree at Shiga University of Medical Science, Japan, and became a doctor of medical science. She is a member of the Department of Obstetrics and Gynecology at the Shiga University of Medical Science and continues to work on reproductive research.
Key message
Dienogest is a fourth-generation progestin possessing anti-androgen activities. We found that it suppressed the activation of primordial follicles and preserved the primordial stockpile and fertility after long-term administration in mice.
Article info
Publication history
Published online: January 15, 2018
Accepted:
December 15,
2017
Received in revised form:
December 15,
2017
Received:
April 5,
2017
Declaration: Shizuka Mita is an employee of Mochida Pharmaceutical Co., Ltd, which sells DNG as a drug. The other authors have no conflicts of interest.
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