A long-term mouse testis organ culture system to identify germ cell damage induced by chemotherapy

Graphical abstract

Highlights

• •Identification of germ cell stages targeted by toxicants using a long-term mouse testis organ culture.
• •Clinically relevant doses of cisplatin damage differentiating spermatogonia.
• •Clinically relevant doses of cisplatin allow recovery from testicular toxicity.
• •The long-term organ culture system serves as an alternative adult testis model.

Abstract

We previously developed the acrosin-green fluorescent protein (GFP) transgenic neonatal mouse organ culture system for rapid and accurate assessment of testicular toxicity. This system effectively evaluates drug-induced toxicity in male germ cells before meiotic entry but cannot assess post-meiotic germ cell toxicity. For many chemicals, the specific stage of germ cell differentiation that is susceptible to toxicity remains unclear, highlighting the need for new methods. In this study, we incubated neonatal mouse testis organ cultures for 35 days to allow post-meiotic cells to develop. The tissue was then exposed to cisplatin to determine the cells that are targeted and to assess the reversibility of the toxicity. We monitored changes in tissue volume and GFP fluorescence, which tracks the progression of spermatogenesis, and confirmed findings by histological analysis. Cisplatin inhibited tissue growth and reduced GFP fluorescence in a concentration-dependent manner. Higher concentrations targeted not only spermatogonia, but also spermatocytes and spermatids. Recovery from toxicity was observed at clinically relevant doses. This study demonstrates that long-term mouse testis organ culture can be used to assess testicular toxicity, enabling the identification of specific germ cell stages targeted by chemicals such as cisplatin.

1. Introduction

Advances in cancer treatment have significantly improved patient survival rates, bringing renewed attention to the long-term consequences of therapies, including their impact on male fertility. However, the treatment benefits of anti-cancer drugs are often given priority over potential long-term side effects. This emphasis means that general toxicity tests tend to be designed with limited dosing or recovery periods, and fertility tests may even be omitted. Consequently, comprehensive data on testicular toxicity and its reversibility may not always be obtained from non-clinical studies. Therefore, there is a growing need to develop methods that can rapidly assess testicular toxicity and new approaches that mimic in vivo intratesticular conditions to effectively evaluate such toxicity.

In mice, spermatogenesis occurs in the seminiferous epithelium of the testis and takes approximately 35 days. The process begins a few days after birth and includes the division of spermatogonial stem cells (SSCs) into spermatogonia, followed by their differentiation into spermatocytes, in which meiosis proceeds. Prophase, the longest of the four meiotic phases, includes preleptotene, leptotene, zygotene, pachytene, and diplotene stages. Subsequently, two sequential rounds of cell division give rise to haploids, namely round spermatids, which undergo dynamic morphological changes, developing into elongating spermatids and finally into spermatozoa. In adults, all these germ cell stages occur in the seminiferous epithelium. However, in neonates, only SSCs and spermatogonia are present in the seminiferous epithelium; spermatocytes and spermatids are absent because meiosis has not yet started (Griswold and Hogarth, 2022).

We previously developed a neonatal mouse testis organ culture system using a polydimethylsiloxane chip on agarose gel (Kojima et al., 2018). This polydimethylsiloxane-ceiling method significantly improves spermatogenic efficiency, enabling long-term evaluation of testicular toxicity and its recovery (Hashimoto et al., 2024, Nakagiri et al., 2024). However, it is challenging to use mature adult mouse testis tissues for in vitro spermatogenesis. While the method used for neonatal tissues can induce spermatogenesis in adult tissue, the efficiency is significantly lower (Sato et al., 2015). Therefore, adult mouse testis tissues are not optimal for toxicity evaluation. Although neonatal tissues may adequately serve as a substitute for adult tissues, there are likely to be inherent differences between them. Additionally, with neonatal tissues, it is impossible to detect toxicity in developing germ cells, such as spermatocytes and spermatids (Hashimoto et al., 2024, Lopes et al., 2021). Nakagiri et al., with the concept similar to ours, incubated tissues for 3 weeks before administering busulfan, that served for preparing the tissue to be stabilized in size expansion and GFP appearance (Nakagiri et al., 2024).

In the present study, we cultured neonatal mouse testis tissues for a pre-incubation period of 5 weeks (35 days), which allowed the tissues to develop differentiating germ cells, including spermatocytes and spermatids. The pre-incubated tissue served as a model for adult tissue to assess the toxicity of cisplatin and enabled the explicit and accurate identification of target cells. This method can be used to detect the target cells of different germ cell differentiation stages that respond to various drugs and chemicals.

2. Materials and methods

2.1. Animals

Double transgenic mice expressing Acrosin (Acr)-green fluorescent protein (GFP) and H3.3-mCherry (Feng et al., 2023) were produced by breeding a female Acr-GFP homozygous mouse (Nakanishi et al., 1999) with a male H3.3-mCherry homozygous mouse (Makino et al., 2014). Both of these lines were occasionally backcrossed with wild-type ICR or C57BL/6 mice for maintenance purposes; therefore, their genetic backgrounds were a mix of ICR and C57BL/6. Testis tissue was obtained from 7-day-old mice. The mice were kept in specific pathogen-free, air-conditioned facilities with a temperature of 24 ± 1 °C and humidity levels of 55 % ± 5 %, under a 13-hour light/11-hour dark cycle. They had ad libitum access to hard food pellets (MF; Oriental Yeast). Their drinking water was acidified to a pH of 2.8–3.0 using HCl. All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation (Animal Research Center of Yokohama City University, Yokohama, Japan; protocol number F–A–23–012).

2.2. Culture method

Testis tissue fragments were cultured on 1.5 % agarose gels that were half soaked in tissue culture medium [α-MEM, supplemented with 40 mg/mL AlbuMAX (11020–021; Thermo Fisher Scientific), 1 × Antibiotic-Antimycotic (GIBCO, #15240096)] as previously described (Hashimoto et al., 2024). A polydimethylsiloxane-ceiling chip with a dent depth of 160 μm (Kojima et al., 2018, Hashimoto et al., 2024, Komeya et al., 2019) was then placed over each tissue fragment. The culture plates were incubated at 34 °C in an atmosphere of 15 % oxygen and 5 % carbon dioxide. The medium was refreshed weekly reaching halfway up the agarose gels. Differences between the current study protocol and our previous protocol (Hashimoto et al., 2024) are illustrated in Fig. 1. In the previous study, the culture incubation period prior to cisplatin exposure was one day. In the present study, testicular tissues from 7-day-old pups were dissected and cultured in vitro for 35 days to allow spermatogenesis to proceed and for germ cells in the seminiferous tubules to differentiate into spermatocytes and spermatids. Following this period, the tissues were exposed to cisplatin, which was designated as exposure day 0 (ED0). Cisplatin was prepared as a stock solution dissolved in aMEM medium at 0.4 mg/mL. This stock solution was added to 2x aMEM medium in which Albumax had been dissolved, in the volume necessary for the desired final concentration, and Antibiotic-Antimycotic and sodium bicarbonate were added, and the mixture was scalded up to a 1x concentration with miliQ. For the cisplatin exposure, media were prepared containing cis-diamminedichloroplatinum (II) (cisplatin; P4394, CAS 15663–27-1, purity ≥ 99.99 %; Sigma Aldrich, St. Louis, MO, USA) at 0, 0.4, 1, 4, 12, and 40 μg/mL, referred to as cis-0, cis-0.4, cis-1, cis-4, cis-12, and cis-40, respectively. The original culture medium was replaced with the new cisplatin-containing media. After exposure to cisplatin for 24 h, the media were replaced with α-MEM containing AlbuMAX, followed by three washes with fresh culture medium at two-hour intervals to eliminate any residual cisplatin. Six testes obtained from three littermate mice were divided into 36 fragments, with six fragments prepared from each testis allocated to each exposure group. Six fragments were observed for GFP analysis (n = 3 fragments) and for histopathology (n = 3 fragments). Post-treatment, testis fragments were monitored for their size and GFP fluorescence levels on ED7, 14, 21, 28, 35, 42, and 49 (n = 3 per group), and histologically evaluated on ED7, 14, 35, and 49 (n = 1 per group), following previously described procedures (Hashimoto et al., 2024).

Fig. 1.

Experimental scheme for evaluating cisplatin-induced testicular toxicity and its reversibility.

2.3. Observation of GFP fluorescence in testes

Cultured testis tissues were examined at least once per week using a stereomicroscope equipped with a GFP excitation light (Leica M205 FA; Leica, Wetzlar, Germany). GFP is expressed in the cytoplasm as the germ cells progressed to the pachytene stage (stage IV) (Hashimoto et al., 2024). Semi-quantitative assessment of Acr-GFP expression was performed by categorizing tissues into six grades based on the GFP-positive area percentage: 0 (0 %), 1 (1 %–20 %), 2 (21 %–40 %), 3 (41 %–60 %), 4 (61 %–80 %), and 5 (81 %–100 %). This grading scale of GFP expression reliably reflects the progression of spermatogenesis (Kojima et al., 2018, Hashimoto et al., 2024). The horizontal projection area of each tissue was measured using Fiji, an open-source software for biological image analysis (Schindelin et al., 2012). To calculate tissue volume, the horizontal projection area was multiplied by the depth of the polydimethylsiloxane-ceiling chip’s indentation. The relative area and volume of each tissue sample were compared with the measurements taken on culture day 0 [CD0 = exposure day − 35; (ED-35)].

2.4. Histological examination of testes

Specimens were fixed with Bouin’s fixative and embedded in paraffin. After thin-sectioning, each specimen was stained with hematoxylin and periodic acid Schiff (PAS) (MUTO PURE CHEMICALS, Tokyo, Japan). The differentiation stage of spermatogenic germ cells was determined based on morphological characteristics, specifically the chromatin pattern within the nucleus and the acrosomal staining profile of PAS-stained images.

2.5. Statistical analysis

Results are presented as the mean ± SE. Statistical analysis was performed using one-way analysis of variance, followed by the Tukey-Kramer honestly significant difference (HSD) test. p < 0.05 was considered statistically significant.

3. Results

3.1. Cisplatin suppresses cultured testicular tissue fragment growth

Every testis tissue fragment was photographed and its volume measured weekly (Fig. 2). Before cisplatin exposure, testis tissue fragments were cultured for a pre-incubation period of 35 days, which were designated as negative days (Fig. 2B). In this period, tissues in all groups showed similar gradual growth and increase in volume, indicating that all groups were comparable at the start of treatment. After cisplatin exposure, tissue volumes began to diverge based on the cisplatin concentration. This divergence highlights the impact of cisplatin exposure, with higher concentrations showing more significant inhibitory effects on tissue growth compared with lower concentrations or the control. In the control group (cis-0), the tissue volume gradually increased, with an approximately two-fold increase at 49 days after exposure (ED49) compared with ED0 (Fig. 2A, B). The tissue volumes in the cis-0.4 and cis-1 groups were also increased to similar extents as the control (Fig. 2A, B). In contrast, tissue fragments exposed to higher concentrations of cisplatin (cis-4, cis-12, and cis-40) exhibited markedly different growth patterns. In the cis-4 group, volume increase was markedly slowed, but after day 21, the tissues seemed to grow at an almost similar rate as those in the control and lower doses groups. This may indicate surviving germ cells whose proliferation and differential activity would be comparable to those in the control and lower doses groups.

Fig. 2.

Changes in testicular tissue volume following cisplatin exposure.

For the cis-12 group, the increase in tissue volume was even more restricted, with the fragments demonstrating minimal growth and eventually plateauing by the later time points (ED35–ED49). Most notably, in the cis-40 group, tissue fragments displayed a sharp decline in growth rate shortly after exposure, with a relatively minor increase in volume that peaked early and remained almost constant or slightly reduced by ED49. This indicates that the highest dose of cisplatin induced severe growth suppression or cytotoxicity that persisted beyond the exposure period.

3.2. Cisplatin represses germ cell differentiation/survival

GFP expression in testis tissue fragments is shown in Fig. 3A. Before cisplatin exposure, all groups showed a steady increase in GFP expression until ED − 21, with no significant differences observed among them (Fig. 3B). Following cisplatin exposure, GFP expression levels began to vary. At ED7 and ED14, GFP levels were similar among the control, cis-0.4, cis-1, cis-4, and cis-12 groups, while the cis-40 group exhibited a marked reduction in GFP expression. By ED21, GFP expression remained comparable among the control, cis-0.4, and cis-1 groups; however, the cis-4 and cis-12 groups showed significantly lower GFP levels compared with the control. Notably, the cis-40 group had completely lost GFP expression by this time. Interestingly, the cis-4 group displayed a significant decrease in GFP expression at ED21 and ED28, followed by partial recovery between ED28 and ED49. This GFP expression recovery corresponds with the tissue volume gain observed in the cis-4 group in Fig. 2B. In contrast, the cis-12 group showed a continuous loss of GFP expression from ED28 onward, resembling the cis-40 group, with no recovery observed.

Fig. 3.

Changes in GFP expression following cisplatin exposure.

3.3. Histological observations of cisplatin toxicity

To corroborate the GFP expression analysis, histological observations were conducted at ED7, ED14, ED35, and ED49 (Fig. 4). In the cis-4 group, the numbers of leptotene and pachytene spermatocytes, as well as spermatids, were comparable with those in the control, cis-0.4, and cis-1 groups at ED7 (Table 1). By ED14, leptotene and pachytene spermatocytes were absent, while spermatids were still present (Table 1), indicating that spermatogonia or preleptotene spermatocytes might have been damaged. By ED35, the germ cell toxicity became evident, as both spermatocytes and spermatids were scarce in the seminiferous tubules. By ED49, spermatocyte numbers had recovered, which was consistent with the GFP expression analysis, indicating that the surviving spermatogonia were repopulating the tissue. In the cis-12 group, spermatids were nearly absent by ED7, and spermatocytes were nearly absent by ED14, indicating that cisplatin could affect both pachytene spermatocytes and spermatogonia/preleptotene spermatocytes. Subsequently, only Sertoli cells remained in most seminiferous tubules at ED35 and ED49, indicating that cisplatin had damaged SSCs. Notably, in the cis-40 group, only Sertoli cells remained in most seminiferous tubules from ED7 onwards, indicating that nearly all germ cell types, including SSCs, were damaged and could not recover from the cisplatin toxicity, as also demonstrated by the GFP analysis. These results indicate that cisplatin toxicity toward germ cells varies in a concentration-dependent manner.

Fig. 4.

Histological evaluation of testicular toxicity following cisplatin treatment.

Table 1.

Presence of germ cell types in mouse testis organ cultures at each time point and cisplatin concentration.

		Cis 0	Cis 0.4	Cis 1	Cis 4	Cis 12	Cis 40
ED7	Elongating spermatid	◯	◯	◯	◯	◯	△
	Round spermatid	◯	◯	◯	◯	◯	△
	Pachytene spermatocyte	◯	◯	◯	◯	◯	✕
	Leptotene spermatocyte	◯	◯	◯	◯	◯	△
ED14	Elongating spermatid	◯	◯	◯	◯	◯	△
	Round spermatid	◯	◯	◯	◯	◯	✕
	Pachytene spermatocyte	◯	◯	◯	✕	✕	✕
	Leptotene spermatocyte	◯	◯	◯	△	△	✕
ED35	Elongating spermatid	◯	◯	△	△	✕	✕
	Round spermatid	◯	◯	△	△	✕	✕
	Pachytene spermatocyte	◯	◯	◯	△	✕	✕
	Leptotene spermatocyte	◯	◯	◯	◯	✕	✕
ED49	Elongating spermatid	◯	◯	◯	◯	✕	✕
	Round spermatid	◯	◯	◯	◯	✕	✕
	Pachytene spermatocyte	◯	◯	◯	◯	✕	✕
	Leptotene spermatocyte	◯	◯	◯	◯	✕	✕

Circles (◯) indicate that germ cells of the specified stage were observed in 10 % or more of the seminiferous tubules. Triangles (△) indicate that germ cells of the specified stage were present but in fewer than 10 % of the seminiferous tubules. Crosses (✕) signify that germ cells of the specified stage were not observed in any seminiferous tubules. Approximately 50 seminiferous tubules (ranging from 24 to 97) were counted for each sample.

4. Discussion

In general toxicity testing, testicular toxicity is routinely evaluated using adult animals. In vitro to in vivo extrapolation is used for rapid regulatory assessment (Bell et al., 2018) and the development of a mouse testis organ culture system that can evaluate testicular toxicity in adult animals would be highly beneficial. However, evaluating testicular toxicity in adults using in vitro systems is highly challenging (Sato et al., 2015). In previously reported mouse organ culture system that uses testis fragments from neonates, cisplatin could only target SSCs and spermatogonia because meiosis had not yet been initiated. Therefore, cisplatin toxicity could not be assessed for other germ cell stages (Hashimoto et al., 2024, Lopes et al., 2021). To overcome this limitation and to gain a more comprehensive understanding of chemotherapy-induced toxicity across all germ cell stages, a novel methodology using a mouse testis organ culture system capable of completing spermatogenesis is required. In this study, we addressed this challenge by developing a long-term in vitro culture system that allows spermatogenesis to be completed before treatment. This system enables the assessment of cisplatin-induced testicular toxicity, including effects on spermatogonia, spermatocytes, and spermatids. Reversibility of toxicity can also be assessed.

The circulating concentration of cisplatin in most cancer patients is between 1–5 μg/mL (Ikeda et al., 1998). Based on GFP measurements, the extent of spermatogenesis in the cis-0.4 and cis-1 groups was almost the same as in the control group. These results indicate that cisplatin did not affect spermatogenesis at concentrations below the clinical dose. We previously demonstrated that the concentration of cisplatin is reduced by half in the culture medium because of its diffusion into the agarose gel (Hashimoto et al., 2024). This indicates that a clinically relevant dose of cis-4 (approximately 2 μg/mL) was potentially toxic, but that the tissue could partially recover. In contrast, cisplatin-induced testicular toxicity did not recover in the cis-12 (6 μg/mL cisplatin exposure) and cis-40 (20 μg/mL cisplatin exposure) groups. These results indicate that cisplatin treatment above clinically relevant concentrations may significantly damage SSCs, preventing long-term recovery of spermatogenesis. In contrast, Lope et al. demonstrated testicular toxicity of cisplatin at doses below the clinical level (0.25, 0.5, 0.75 μg/mL) using a mouse testis organ culture system (Lopes et al., 2021). However, in our present and previous studies, cis-1 (approximately 0.5 μg/mL) did not exhibit apparent testicular toxicity (Hashimoto et al., 2024). This discrepancy may be attributed to methodological differences. One difference is that Lope et al. used germ cell markers such as PLZF and SCP3 to assess testicular toxicity, whereas we primarily relied on Acr-GFP expression. Another is that they placed testis tissue fragments on a polyacrylamide membrane, whereas we used an agarose gel as a tissue stand and a PDMS chip to cover the tissue.

The results presented here correlate well with previous findings in animal experiments. Indeed, a 10 mg/kg i.p. or i.v. administration of cisplatin to mice can affect differentiating spermatogonia, spermatocytes and spermatids, whereas a 1 mg/kg i.p. or i.v. administration affects only differentiating spermatogonia (Meistrich et al., 1982). The circulating concentration of cisplatin reaches a maximum of 10 μg/mL after a single 10 mg/kg i.v. administration (Lancaster et al., 2013). Importantly, the LD₅₀ of cisplatin dissolved in saline is approximately 15 mg/kg i.p., primarily because of nephrotoxicity (Mannel et al., 1989), meaning that circulating concentrations of cisplatin do not exceed 15 μg/mL. Therefore, cis-40 (approximately 20 μg/mL) exposure cannot occur in vivo.

In the present study, GFP expression in the control group gradually decreased from ED14 (49 days of culture) with some ongoing fluctuation. This may be a limitation of this in vitro system for evaluating the reversibility of testicular toxicity over time. Further development is needed to refine this in vitro method for evaluating testicular toxicity and its reversibility and to maintain spermatogenesis for extended periods so as to mimic in vivo conditions. Additionally, although we used double transgenic mice harboring both Acr-GFP and H3.3-mCherry, we omitted mCherry as a toxicity marker in this study. While mCherry appears in elongating spermatids (step 11 or beyond), its expression varies between tissues and time points, making it unreliable for toxicity evaluation. In contrast, Acr-GFP exhibited greater stability across experiments, leading us to use it as the primary marker for monitoring spermatogenesis. The variability in mCherry expression is not entirely understood but appears to be inherent to in vitro conditions. One possible factor is the lack of fluid flow in the seminiferous tubules, which contributes to a less consistent microenvironment compared to in vivo conditions. Additionally, detecting mCherry requires high magnification with an inverted microscope at the end of each culture period. Despite these limitations, mCherry could still be a valuable marker for assessing the final stages of spermatogenesis in future studies, particularly with more robust culture methods.

Adult animal models present challenges in evaluating chemotherapy-induced testicular toxicity and its reversibility; however, this study demonstrates that our novel alternative method can overcome these limitations.

Funding

This study was supported by a JST CREST grant (No. JPMJCR21N1 to TO), a JSPS KAKENHI Grant-in-Aid (no. 22H00485 to TO), and the Japan Agency for Medical Research and Development (grant no. 24gn0110086h0001 to TO and grant no. 24mk0121304j0001 to SY).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.