Induction of Meiotic Initiation in Long-Term Mouse Spermatogonial Stem Cells Under Retinoid Acid and Nutrient Restriction Conditions

Abstract

Spermatogonial stem cells (SSCs) produce haploid sperm via mitosis and meiosis in vivo. Although the technique to culture mouse SSCs has been well established, induction of meiosis in vitro has remained a challenge. Retinoic acid (RA) is required for meiosis in vivo; however, RA alone is not sufficient to induce meiosis in vitro. Here, we describe a method in which nutrient restriction and RA synergistically induce meiotic initiation into meiotic prophase I in cultured mouse SSCs.

1. Introduction

The long-term culture of stem cells with germline potential has broad applications for advancing basic science research, fertility preservation, and regenerative medicine. In addition to spermatogonial stem cells (SSCs) [1], recent progress in the derivation of primordial germ cell-like cells (PGCLCs) from pluripotent stem cells (PSCs) has also provided an alternative source of germ stem cells (GSCs) [2]. Similar to embryonic stem (ES) cell culture, GSCs are usually seeded onto a monolayer of supporting cells (e.g., STO cells, mouse embryonic fibroblasts or MEFs) that provide essential nutrients and growth factors [3]. Alternatively, male GSCs are cultured in defined culture media, optimized to support their maintenance and proliferation. These media typically contain spe-cific growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF or FGF2), which are known to promote the survival and proliferation of male GSCs [4, 5].

SSCs are capable of self-renewal to maintain the stability of GSCs and differentiation to produce mature spermatozoa through meiosis. Meiosis is a defining feature of germ cells, during which replicated parental chromosomes undergo homologous recombi-nation during prophase I, followed by two rounds of divisions to halve chromosome contents [6].

Despite the progress in culturing GSCs in vitro, induction of meiosis in cultured SSCs has remained a challenge [7]. It is widely known that retinoic acid (RA) is required for meiosis in vivo, in that meiosis does not initiate and spermatogenesis stalls at the stage of undifferentiated spermatogonia in mice fed with vitamin A-deficient (VAD) diet [8] or administrated with compounds, e.g., WIN 18 446, that inhibit retinaldehyde dehydrogenases to block RA production from retinal [9]. However, RA alone is not sufficient to induce meiosis in vitro under normal culture condition [10] or in vivo [11]. Thus, meiotic induction usually requires transplantation of culture SSCs back to seminiferous tubules.

In our laboratory, we found that STRA8, an essential gate-keeper of meiotic initiation downstream of RA, acts as a suppressor of autophagy and that Stra8-deficient germ cells fail to initiate meiosis showing aberrant autophagy activation [12]. These results revealed for the first time a link between autophagy, a protein and cellular organelle recycling process through lysosomes [13], and the beginning of meiosis in mammalian male germ cells. Interest-ingly, nutrient restriction, probably the most potent inducer of autophagy [14], initiates meiosis in yeasts [15, 16]. On this basis, we devised and describe here in detail a simple culture system in which nutrient restriction and RA synergistically induce meiotic initiation in cultured mouse SSCs, faithfully recapitulating the tran-scriptomic and cytological features of the first stages of meiotic prophase I occurring in vivo [10].

2. Materials

2.1. Establishment of Long-Term Mouse SSC Culture

1. 3–4 male mice in C57BL/6 X DBA/2 F1 hybrid background at postnatal days 6–8 (see Note 1).

2. Sterile 70% ethanol.

3. Sterilized surgical tools (sharp scissors, forceps, spring scissors, etc.).

4. Sterile 1.5-mL microcentrifuge tube.

5. Sterile 15 and 50 mL polypropylene conical tubes.

6. Phosphate buffered saline (PBS) (pH 7.4) (Gibco Catalog No. 10-010-072).

7. Hanks’ Balanced Salt Solution (HBSS) (no calcium, no mag-nesium, no phenol red) (Gibco Catalog No. 14175-095).

8. Microcentrifuge, preferably with temperature control, to pro-cess samples at 4 °C.

9. CO₂ incubator.

10. Collagenase, Type IV (Gibco Catalog No. 17104-019). Freshly prepared as 2 mg/mL solution in HBSS on the day of use.

11. Deoxyribonuclease I from bovine pancreas (DNase I) (Sigma DN25). Prepared as 2 mg/mL solution in HBSS. Kept frozen until use.

12. Trypsin-EDTA (0.25%) (Gibco Catalog No. 25200-056).

13. Cell strainer (40 μm pore size) (Corning Catalog No. 352340).

14. Cell counter (Invitrogen Countess II).

15. Tissue culture dishes (vacuum-gas plasma tissue-culture trea-ted polystyrene, 60 mm).

16. 0.1% gelatin solution (ATCC Catalog No. PCS-999-027).

17. Mitotically arrested mouse embryonic fibroblasts (MEFs) (see Note 2).

18. Media for MEF cells: DMEM (Gibco 11995-065), Fetal bovine serum (FBS) Gibco A3160501).

19. SSC media (see Table 1).

Table 1.

Components of SSC media

	Company	Catalog number	SSC media
IMDM	Thermo Fisher	12440-053
N2	R&D Systems	AR-009	1X
Pyruvic acid	Sigma	P2256	200 μg/mL
D-(+)-glucose	Sigma	G7021	6 mg/mL
DL-Lactic acid	Sigma	L4263	1 μL/mL
Bovine serum albumin (BSA)	MP Biomedicals	810661	5 mg/mL
L-Glutamine	Sigma	G7513	2 mM
2-Mercaptoethanol	Sigma	M3148	5 × 10−5 M
MEM vitamin	Thermo Fisher	11120-052	1X
NEAA	Thermo Fisher	11140-050	1X
Ascorbic acid	Sigma	A4544	10−4 M
d-Biotin	Sigma	B4501	10 μg/mL
β-Estradiol	Sigma	E2758	30 ng/mL
Fetal bovine serum	Hyclone	SH30396.03	10 μL/mL
KSR	Thermo Fisher	10828-028	50 μL/mL
Human FGF2	Peprotech	100-18B	10 ng/mL
Rat GDNF	Peprotech	450-51	15 ng/mL

2.2. Meiotic Induction Media

1. Earle’s Balanced Salt Solution (EBSS), calcium, magnesium, phenol red (Gibco Catalog No. 24010-043).

2. 0.1 mM all-trans retinoic acid (Sigma, R2625) dissolved in DMSO.

2.3. Meiotic Progression Media

1. Stock solutions: 10⁻⁴ M melatonin (Sigma M5250) in DMSO; 10⁻¹ M 5α-Dihydrotestosterone (DHT) (APExBIO B8214) in DMSO; Bovine pituitary extract (BPE) (Gibco 13028-014); 200 μg/mL follicle-stimulating hormone (FSH) (Sigma F4021) in IMDM.

3. Methods

3.1. Establishment of Mouse SSC Culture

1. Euthanize 3–4 mouse male pups under CO₂.

2. Testes are collected in a 60 mm dish containing 2 mL sterile HBSS using the aseptic technique on ice.

3. After the removal of tunica albuginea, testes are cut into small pieces using spring scissors.

4. Tissues are transferred to a 15 mL tube by using a transfer pipette containing 2 mL of 2 mg/mL Collagenase IV solution (final collagenase IV concentration: 1 mg/mL). The tubules are gently dispersed by pipetting.

5. Tissues are incubated in 37 °C water bath for 20 min until tissues are separated (gently pipetting if necessary).

6. Tubule fragments are washed with the addition of 4 mL of HBSS, followed by centrifugation at 300× g for 5 min at 4 ° C. The supernatant is removed.

7. Tubule fragments are incubated in 1 mL of 0.25% trypsin-EDTA solution, 1 mL of HBSS, and 2 mL of 2 mg/mL DNase I in 37 °C water bath for 5 min.

8. After incubation, 1 mL of FBS is added to neutralize trypsin.

9. Gently pipette the mixture to generate a single-cell suspension.

10. Pass the cell suspension through a 40-μm cell strainer.

11. Collect cells by centrifugation at 300× g for 5 min at 4 °C. Discard the supernatant.

12. Add 4 mL of HBSS to the cell pellet. Wash cells by gentle pipetting.

13. Collect cells by centrifugation at 300× g for 5 min at 4 °C. Discard the supernatant.

14. Resuspend testicular cells in 4 mL of SSC media. Measure cell concentration by cell counter.

15. Plate 1 × 10⁶ testicular cells on 0.1% gelatin-coated 60 mm dish with 4 mL SSC media. Incubate overnight in CO₂ incubator at 37 °C.

16. Plate MEFs in 60 mm dishes in 4 mL of DMEM containing 10% FBS at 70–80% confluency. The number of MEFs used to achieve this confluency should be empirically determined.

17. The next day, non-adherent cells (germ cells) are collected into 15 mL conical tubes. Cells are collected by centrifugation at 300× g for 5 min at 4 °C. The supernatant is discarded. Adherent cells (somatic cells, e.g., Sertoli cells) are discarded. Based on our experience, this procedure—differential plating [17]—is an effective step to enrich SSCs.

18. Remove media from MEF feeder cells.

19. Resuspend the collected non-adherent cells (germ cells) in 4 mL of SSC media. Plate the cells on MEFs.

20. Change media every other day by removing all media from plates using gentle pipetting, followed by adding 4 mL of fresh SSC media.

21. When SSCs reach 70–80% confluency, split the cells by stan-dard cell culture procedure. Plate cells onto new MEFs.

3.2. Induction of Meiotic Initiation

1. To induce meiotic initiation, 1.0 × 10⁶ cells from SSC culture are plated on 60-mm dishes coated with MEF feeder cells (see Note 3).

2. Next day, remove SSC media and add 4 mL of meiotic induction media. Meiotic induction media is composed of 10% of SSC media, 90% EBSS (e.g., 1 mL of SSC media and 9 mL of EBSS), and 100 nM retinoic acid (1:1000 dilution from the stock solution) (see Note 4).

3. Incubate SSCs in meiotic induction media in CO₂ incubator at 34 °C for 2 days (see Notes 5 and 6).

4. At this time, early meiotic events, such as activation of key meiotic genes and DNA double-strand breaks (DSBs), can be detected as reported (Fig. 1) [10].

Fig. 1.

Nutrient restriction synergizes with RA to induce meiotic initiation in SSC culture. (a) Bright-field images of SSC culture with indicated treatments for 2 days. Scale bars, 50 μm. (b) Relative expression of Spo11, Dmc1, and Sycp3 against Gapdh analyzed by qRT-PCR in SSC culture with indicated treatments for 2 days. (c) Immunostaining for γH2AX (red) and DAPI (blue) with indicated treatment for 2 days. Scale bars, 10 μm. (d) Immunostaining for DMC1 (red), DDX4 (green), and DAPI (blue) in Spo11^+/+ and Spo11^−/− SSC culture. Scale bars, 10 μm. In panels c and d, the numbers below the immunofluorescent images indicate the percentage of cells seen with the relevant staining under different conditions

3.3. Meiotic Progression

1. 1. To further promote meiotic progression through leptonema and up to the zygotene stage, replace meiotic induction media with meiotic progression media. Meiotic progression media is composed of SSC media (Table 1) without GDNF and FGF2 supplemented with 10⁻⁷ M melatonin, 10⁻⁶ M DHT, 50 ng/mL BPE, and 200 ng/mL FSH (see Note 7).

2. Incubate cells with meiotic progression media at 34 °C for 4 days (see Notes 5 and 6).

3. At this time, prophase I leptotene and zygotene events, such as the formation of the lateral and central elements of the synaptonemal complex, can be examined as reported [10] (Fig. 2) (see Note 8).

Fig. 2.

Nutrient restriction and RA-induced meiotic initiation supports meiotic progression. (a) DAPI staining and immunostaining for SYCP1 (red), SYCP3 (green), and VASA (cyan) in SSC cultures with indicated treatment. Scale bars, 10 μm. (b) Representative chromosome spreads stained by SYCP1 (red) and SYCP3 (green) from SSC cultures following the indicated treatment are shown

4. Notes

1. The genetic background of donor mice has a significant influ-ence on the establishment of mouse SSC culture. In our experience, testicular cells from either C57BL/6 X DBA/2 F1 hybrid background or CD1 background produce SSC cultures with rapid growth, while testicular cells from C57BL/6 inbred background do not sustain in vitro growth after 1 month. The age of the donor mouse pups is preferentially to be between 6- and 8 days of age. In a typical preparation, testes from 3 to 4 pups are used. Testes from fewer animals are also able to establish SSC culture, but smaller dishes, e.g., 35 mm dishes, should be used for the initial culture.

2. Commercially available MEF cells, e.g., R&D system Catalog No. AR005, are used. According to the manufacturer’s website, MEF cells are collected from outbred CF1 mice, mitotically arrested by irradiation.

3. MEF cells may produce cytokines that inhibit cell differentiation. Use MEF cells at lower density.

4. Reducing the concentration of FGF2 and GDNF with RA treatment is not sufficient to induce meiosis. FGF2/GDNF are not inhibitors of meiosis and cultured SSCs do not spontaneously initiate meiosis without FGF2 and GDNF. This is consistent with past literature, which has shown that the targets of GDNF and FGF2 are mostly related to cell cycle regulators and spermatogonial stem cell self-renewal, but not directly related to meiosis [18–21]. Moreover, in F9 embryonic carci-noma cells that do not require GDNF and FGF2 in the culture system, RA treatment under nutrient restriction conditions was sufficient to induce meiotic initiation, suggesting that this process does not involve GDNF and FGF2.

5. If the density of cultured SSCs is high, refresh media every day.

6. It is known that temperature plays a role in male germ cell differentiation in vivo. Culturing cells at 34 °C, rather than 37 ° C, can greatly improve cell survival under meiotic induction media and meiotic progression media.

7. RA is not used in the whole experiment: RA is used only in meiotic induction media but not in the meiotic progression media.

8. Instead of using trypsin to harvest cultured cells, TIM buffer [22] mixed with 1 mg/mL collagenase IV can be used to directly harvest cells by gentle pipetting and subsequent chro-mosome spread analysis.