Ovarian and follicular culture enables the production of fertile oocytes in C57BL/6 mice

Abstract

In vitro experiments have become a powerful alternative to in vivo approaches for analyzing gene functions and molecular mechanisms. However, successful in vitro oogenesis using the C57BL/6 strain–widely used as a standard mouse model in mammalian studies–has not yet been reported. In this study, we established a 30-day culture system combining ovarian and follicular culture, and successfully produced fully competent oocytes from neonatal C57BL/6N ovaries, which contain only non-growing oocytes prior to primordial follicle formation. A key contributing factor to this success was the supplementation of dibutyryl cyclic AMP, a membrane-permeable analogue of endogenous cyclic AMP, which promoted follicular growth. This culture system provides a valuable tool for investigating the mechanisms underlying oogenesis.

In vitro systems provide powerful tools for analyzing mammalian developmental processes. In mice, protocols for in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture of cleavage–stage embryos (IVC) are well established, enabling detailed investigations into the metabolic and molecular mechanisms underlying oocyte maturation and early embryogenesis [1, 2]. In addition, in vitro gene knockdown approaches have facilitated the analysis of zygotic gene activation and the maternal-to-zygotic transition without generating knockout animals [3, 4]. Although in vitro oocyte growth (IVG) systems can now produced fully functional oocytes from non-growing oocytes or primordial germ cells, most successful studies have been confined to hybrid mouse strains, which are more robust in vitro [5,6,7,8]. In contrast, inbred strains such as C57BL/6 (B6) are highly sensitive to in vitro manipulation [9,10,11,12,13,14,15,16,17], limiting their use despite their importance as standard genetic models [18]. Establishing a reliable IVG system for B6 mice would therefore greatly expand experimental opportunities for studying maternal factors. In this study, we explored culture conditions suitable for IVG in B6 mice.

The only method for the successful in vitro production of functionally mature oocytes from neonatal mouse ovaries consists of three steps: ovarian culture, follicular culture, and IVM of cumulus–oocyte complexes (COCs) [5, 6, 19]. The insert membrane used for ovarian culture is crucial for maintaining three-dimensional ovarian growth and enabling follicle isolation. Because the Transwell-COL PTFE insert membrane (Cat# 3492; Corning) previously used for ovarian culture is no longer commercially available [5,6,7, 19,20,21], we evaluated several alternative membranes. Among four membranes tested, fetal ovarian cells cultured on PET or PC membranes appeared flattened and dispersed whereas those cultured on Millicell PTFE membrane retained better organization. Secondary follicles were observed only on the Millicell PTFE membrane in fetal ovarian culture (Supplementary Fig. 1). In neonatal ovarian culture, secondary follicles were formed on all membranes (Supplementary Fig. 2); however, follicles and oocytes were more clearly visualized on the Millicell PTFE membrane. Although ovarian explants tended to detach from this membrane during follicle isolation, coating with collagen I/III and iMatrix, a recombinant laminin-based extracellular matrix, improved tissue adhesion. Based on these results, the Millicell PTFE membrane was selected.

Estrogen signaling can disrupt normal follicle assembly [22, 23]. While neonatal ovarian culture has been successfully performed without estrogen receptor antagonists, such as ICI 182,780 (ICI), in several mouse strains [5, 6, 19], strain-specific differences in estrogen sensitivity have been reported. In particular, ovaries from B6 mice exhibited higher estrogen receptor sensitivity than those from other commonly used strains [24]. Therefore, we examined the effects of estrogen receptor inhibition. In neonatal B6 ovarian explants, treatment with 5 µM ICI during days 0–6 significantly increased the number of secondary follicles isolated after 15 days of culture compared with ICI-free conditions, whereas treatment during days 0–4 did not result in a significant improvement (Supplementary Fig. 3A). Immunostaining for laminin further supported these findings. In ovaries treated with ICI during days 0–6, laminin formed a continuous envelope surrounding the granulosa cell layers, within which a single oocyte was enclosed. Under ICI-free conditions, laminin and granulosa cell organization were incomplete, and multiple oocytes were enclosed within them (Supplementary Fig. 3B). Thereafter, ovarian culture was performed using ICI during days 0–6 of the total 15-day culture.

Despite improved follicle assembly, histological analysis revealed limited follicular growth in B6 ovaries (Fig. 1A). A previous study showed that culturing primary follicles isolated from B6 × CBA/2 hybrid mice in a medium containing dibutyryl cyclic AMP (dbcAMP) significantly increased follicle and oocyte size, as well as the maturation rate to the MII stage [25]. To enhance follicular development, we examined the effects of dbcAMP, a membrane-permeable analog of cyclic AMP (cAMP). cAMP is a key intracellular messenger synthesized from ATP by membrane-associated adenylate cyclase and degraded by intracellular phosphodiesterase. It activates protein kinase A (PKA), which phosphorylates cAMP response element-binding protein (CREB) and induces the transcription of genes involved in granulosa cell proliferation and differentiation [26]. Supplementation of 60 µM dbcAMP during ovarian culture significantly increased both the number of isolated secondary follicles, as well as the diameters of the oocytes and follicles in B6 ovaries (Fig. 1B) [27]. Similar effects on follicle number and size were observed in B6 × DBA/2 hybrid (B6D2F1) ovaries (Fig. 1B), although oocyte size was unaffected [27].

Fig. 1.

Effects of dbcAMP treatment on secondary follicle formation in neonatal ovarian explants. (A) Sequential changes in neonatal B6 ovaries cultured in the absence or presence of dbcAMP. Bars; 200 μm. (B) The numbers of isolated secondary follicles per ovary and the diameters of isolated secondary follicles and oocytes cultured in the absence or presence of dbcAMP on day15. N. S. indicates no statistically significant difference.

We next evaluated dbcAMP supplementation during follicular culture. In B6 mice, a significantly greater number of COCs developed in the dbcAMP-treated group compared to the untreated group by day 14 of follicular culture following ovarian culture with dbcAMP (Table 1 and Fig. 2). In contrast, even without dbcAMP during both ovarian and follicular culture, COC formation in B6D2F1 mice was significantly higher than that in any B6 group, indicating marked intrinsic strain differences. In addition, more than 70% of secondary follicles isolated from juvenile B6 mice formed COCs under dbcAMP-free follicular culture conditions (Table 1), indicating that the ability of in vivo-derived secondary follicles to form COCs was much higher than that of secondary follicles derived from neonatal ovarian explants.

Table 1. Developmental competences of in vitro-produced oocytes.

Strain (age of donors)	Conditions of ovarian culture			Conditions of follicular culture			No. of
	ICI days 0–6	dbcAMP days 0–15	O2	collagenase treatment	dbcAMP days 0–14	O2	ovaries	isolated secondary follicles	COCs	MII oocytes	normally fertilized eggs	2-cell	offspring
B6 (P0)	+	–	20%	day 8	–	7%	6	67	10	8	2	1	0
									(14.9%) c	(80%)	(25.0%)	(50.0%) abc	(0%)
B6 (P0)	+	+	20%	day 8	–	7%	6	177	44	41	20	14	0
									(24.9%) c	(95%)	(48.8%)	(70.0%) c	(0%)
B6 (P0)	+	+	20%	day 8	+	7%	6	189	87	79	34	33	7
									(46.0%) b	(90.8%)	(43.0%)	(97.1%) ab	(21.2%)
B6 (P0)	+	+	20%	day 8	+	20%	6	189	84	74	33	28	5
									(44.4%) b	(88.1%)	(44.6%)	(84.8%) abc	(17.9%)
B6D2F1 (P0)	+	–	20%	day 8	–	7%	6	133	94	86	53	52	11
									(70.7%) a	(91.5%)	(61.6%)	(98.1%) a	(21.2%)
B6 (P12)	N.A.	N.A.	N.A.	day 0	–	7%	21	160	115	108	62	59	9
									(71.9%) C	(93.9%) B	(57.4%) A	(95.2%)	(15.3%)
FVB (P12)	N.A.	N.A.	N.A.	day 0	–	7%	16	117	98	90	28	23	5
									(83.8%) B	(91.8%) B	(31.1%) B	(82.1%)	(21.7%)
B6D2F1 (P10)	N.A.	N.A.	N.A.	day 0	–	7%	8	129	128	128	67	57	11
									(99.2%) A	(100%) A	(52.3%) A	(85.1%)	(19.3%)

Different letters indicate significant differences. N.A. indicates “Not Applicable”.

Fig. 2.

Developmental competence of in vitro-produced oocyrtes from ovaries of B6 neonates. (A) In vitro-produced oocytes from neonatal B6 ovaries and their developmental fate after IVM, IVF, and embryo transfer. 2PN indicates normally fertilized eggs in which two pronuclei are formed. Bars; 100 μm. (B) Analysis of microsatellite markers distinguishing offspring derived from B6 and B6D2F1 mice. Bacause B6 and B6D2F1 mice have the same hair and eye color, the genetic origin of offspring derived from in vitro-produced oocytes was confirmed using microsatellite markers, demonstrating the successful production of oocytes with full developmental competence.

To assess the functional competence of oocytes produced under these conditions, IVG, IVM, IVF, IVC, and embryo transfer experiments were performed. In B6, oocytes produced without dbcAMP during both ovarian and follicular culture or during follicular culture alone exhibited poor developmental competence, and no offspring were ultimately bone (Table 1). In contrast, dbcAMP supplementation during both ovarian and follicular culture enabled B6 oocytes to develop to the 2-cell stage and produce offspring following embryo transfer, irrespective of whether follicular culture and subsequent in vitro procedures were performed under 7% or 20% O₂, (Table 1 and Fig. 2) [27]. Although a 7% O₂ environment has previously been shown to enhance blastocyst development, in the present study, development to the blastocyst stage (76.5% vs. 51.4%) and to term (21.2% vs. 17.9%) tended to be higher under a 7% O₂ than under 20% O₂, but these differences were not statistically significant. Finally, 2-cell embryos produced in vitro from secondary follicles isolated from juvenile B6, FVB, and B6D2F1 mice without dbcAMP supplementation were competent to develop into offspring following embryo transfer. Collectively these results demonstrate that when neonatal B6 ovaries are used as starting material, dbcAMP supplementation during both ovarian and follicular culture is critical for producing developmentally competent oocytes.

The requirement for dbcAMP in producing functionally mature oocytes specifically from the ovaries of B6 neonatal mice may be attributed to insufficient endogenous cAMP levels. This insufficiency could result from ATP depletion, reduced adenylate cyclase activity, or increased phosphodiesterase activity during ovarian and subsequent follicular culture. Therefore, not only dbcAMP but also phosphodiesterase inhibitors might enable the in vitro production of functionally mature oocytes from the ovaries of B6 neonatal mice. Furthermore, in B6D2F1 mice, oocyte size was unchanged, whereas follicular size differed between dbcAMP-treated and conventionally cultured ovaries, suggesting that dbcAMP primarily affected granulosa cells. cAMP regulates transcription through the PKA–CREB axis in oocytes and granulosa cells within antral follicles, including genes involved in cell cycle progression, glycolysis, mitochondrial function, steroidogenesis, and other related process [26, 28]. Although the precise effects of cAMP in oocytes and granulosa cells during the growth stage remain unclear, dbcAMP-induced transcriptional changes in granulosa cells may enhance their proliferation and differentiation, thereby promoting oocyte growth and COC development.

B6-derived cells and embryos are highly sensitive to in vitro conditions. Most blastocysts produced through IVF and IVC lose full-term developmental potential after embryo transfer despite only four days of culture [15, 16]. In this study, B6 oocytes and ovarian somatic cells were cultured for 31 days, yet dbcAMP likely mitigated their fragility. These findings suggest that fragility is not solely determined by culture duration but may involve stage-specific mechanisms, and that there may still be room for further optimization of IVF and IVC conditions for B6 mice.

In conclusion, to the best of our knowledge, we successfully produced functionally mature oocytes from neonatal mouse ovaries in vitro for the first time in the standard inbred strain, B6. In the near future, our in vitro system will contribute to understanding the mechanisms underlying oogenesis, from primordial follicle assembly to the development of fully functional mature oocytes.

Materials

Animals

C57BL/6NJcl, DBA/2, FVB/NJcl, and B6D2F1 mice (CLEA Japan Inc., Tokyo, Japan) were used for the IVG and IVF experiments. Jcl:ICR mice (CLEA Japan Inc.) were used to prepare recipients for embryo transfer. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Tokyo University of Agriculture (approval numbers: 240111).

Collection of ovaries, follicles, and sperm from mice

In the preliminary experiments, B6D2F1 fetal ovaries were harvested at embryonic days 12.5 from B6 female mice that had been mated with DBA/2 males. For main experiments, ovaries were collected from B6D2F1 and B6 neonatal female mice on postnatal day (P)0. Secondary follicles were isolated from juvenile female mice of the B6D2F1, B6, and FVB strains at P10, P12, and P12, respectively as previously described [29, 30]. Spermatozoa were collected from the cauda epididymis of adult male mice of the B6D2F1, B6, and FVB strains between 10 and 24 weeks of age.

Preliminary evaluation of insert membranes for ovarian culture

To explore the most suitable insert membrane for ovarian culture, we evaluated the Millicell PTFE insert membrane (Cat# PICM0RG50; Merck Millipore, Darmstadt, Germany), the Cell culture insert PET membrane (Cat# 353092; Falcon, Corning, Corning, NY, USA), the ThinCert PET membrane (Cat# 657630; Greiner Bio-One, Kremsmünster, Upper Austria), and the Transwell PC insert membrane (Cat# 3414; Corning).

The α-MEM (Gibco, Waltham, MA, USA) was prepared by adding 1.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (Tokyo Chemical Industry, Tokyo, Japan) and 10 units/ml penicillin–10 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and is hereafter referred to as the α-MEM. This medium, further supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma-Aldrich), was used as the conventional ovarian culture medium. Fetal and neonatal ovaries from B6D2F1 fetuses or mice were cultured on each insert membrane in 1820–2020 µl of conventional ovarian culture medium under gas–liquid interphase conditions for 17 and 10 days, respectively, at 37°C in an atmosphere of 5% CO₂ and 95% air. Half of the medium was replaced every other day.

Ovarian culture

Prior to ovarian culture, each Millicell insert membrane (Cat# PICM0RG50; Merck Millipore) was coated with 500 µl of a collagen solution, prepared by adding 50 µl of collagen I solution (Cat# IPC-50; KOKEN, Tokyo, Japan) and 84 µl of collagen III solution (Cat# PSC-3-100-20; Nippi Inc., Tokyo, Japan) to 366 µl of ethanol. The final concentrations of both collagen I and collagen III were 0.5% (w/v). After coating, the membranes were air-dried overnight. If not used immediately, the coated membranes were stored at 4°C, protected from light, for up to two months. Immediately before use, 1.6 µl of iMatrix-511 (Cat# 892-011; Nippi Inc.) was dissolved in 1500 µl of phosphate-buffered saline (PBS), and the entire volume was applied to each membrane. One hour later, the supernatant was removed, and the membranes were washed with PBS.

The effects of 5 µM ICI (Tocris, Minneapolis, MN, USA) and 60 µM dbcAMP (Sigma-Aldrich) on follicular formation and growth were examined. ICI was added to the conventional medium during either days 0–4 or days 0–6 to promote follicular formation. Follicular growth was assessed in the presence or absence of dbcAMP. Four to six ovaries were cultured on a single Millicell insert membrane in 1820 µl of the respective ovarian culture medium for 15 days at 37°C in an atmosphere of 5% CO₂ and 95% air. Half of the medium was replaced every other day [21].

Follicular culture

Secondar follicles were isolated from ovarian explants on day 15 in L15 medium (Sigma-Aldrich) supplemented with 0.4% (w/v) polyvinylpyrrolidone (PVP, molecular weight = 360,000, Sigma-Aldrich) using tungsten needle (Cat# 10130-20; Muromachi Kikai, Tokyo Japan). The α-MEM supplemented with 2% (w/v) PVP, 5% (v/v) FBS, and 0.1 IU/ml FSH (FOLLISTIM Injection 50; MSD, Tokyo, Japan) was used as the conventional follicular culture medium. The effects of 60 µM dbcAMP and oxygen concentrations were investigated. Follicular growth and viability was assessed in the presence or absence of dbcAMP [21]. The developmental competence of oocytes produced by ovarian culture under 20% O₂, followed by follicular culture under either 7% or 20% O₂ was compared [29]. Forty to sixty follicles were cultured on a single Millicell insert membrane in 3 ml of the respective follicular culture medium for 14 days. Secondary follicles isolated from ovarian explants were treated with 0.1% (w/v) collagenase type I (Worthington Biochemicals, Lakewood, NJ, USA) at 37°C for 25 min on day 8 after the initiation of follicular culture. After washing, the follicles were cultured on Millicell membranes coated with collagen types I and III, but not with iMatrix, as described above. Previous studies have shown that collagenase treatment of secondary follicles derived from cultured ovaries, when applied immediately after follicle isolation, reduced their viability subsequent follicular culture [20]. Therefore, in the present study, collagenase treatment of the resulting secondary follicles was performed on day 8 of follicular culture. Prior to collagenase treatment, uncoated Millicell membranes were used. Half of the medium was replaced every other day [21].

Secondary follicles were isolated from juvenile mice of each strain using 27G needle (Nipro, Osaka, Japan) in L15 medium supplemented with 0.4% (w/v) PVP. The follicles were treated with 0.1% (w/v) collagenase type I. After washing, they were cultured on uncoated Millicell membranes with 3 ml of the conventional follicular culture medium for 14 days at 37°C in an atmosphere of 5% CO₂, 7% O₂, and 88% N₂. Half of the medium was replaced every other day [29].

Statistical analysis

All experiments were performed at least three times. Statistical significance in the number of isolated secondary follicles was assessed using Dunnett’s post hoc test following one-way ANOVA for ICI treatment. Student’s t-test was used for pairwise comparison assuming equal variances to evaluate the yield of secondary follicles per ovary following dbcAMP treatment. Fisher's exact test with Benjamin–Hochberg adjustment was applied for multiple group comparisons of developmental competence. P values less than 0.05 were considered statistically significant.

Methods for IVM, IVF, IVC, embryo transfer, PCR analysis, immunostaining, and histology are described in the Supplemental Text. A summary of protocols for in vitro production of fertile oocytes from neonatal or juvenile B6 mice is shown in Supplementary Fig. 4.

Conflict of interests

The authors declare no conflict of interest.

Funding Statement

This study was supported by KAKENHI (grant numbers; 18H05547, 23K27088, and 25H01353).