A Novel Approach of Tracing in Vivo Bioluminescence Imaging Expression of Vitrified Immature Testicular Tissue Grafts Until Adulthood: A Translational Transgenic Mouse Model


 Background: The optimal method for cryopreserving immature testicular tissue (ITT) remains unknown and there is no standardized protocol. Controlled slow freezing remains the mainstream method of choice in human prepubertal male fertility preservation. Currently, the outcomes for ITT vitrification are conflicting, and most data are limited to in vitro animal studies.Methods: A total of 12 pairs of donor and recipient mice were included in our experiments. The donors were immature transgenic mice, and the recipients were wild-type male mice. In the vitrification group, ITT was vitrified and thawed before transplantation. In the control group, ITT was transplanted to the recipients immediately. After thawing, we measured the expression of apoptosis-related mRNA caspase-3. More importantly, we monitored to adulthood all the transplanted grafts in vivo using noninvasive bioluminescence imaging (BLI) technology. On day 31, we removed the grafts for evaluation via hematoxylin and eosin staining and immunohistochemistry (IHC).Results: We traced the survival of the grafts by in vivo BLI on days 1, 2, 5, 7, and 31 after transplantation. In both the vitrification and the control groups, bioluminescence decreased between days 2 and 5. Subsequently, the bioluminescence showed an upward trend until day 31. Compared with day 1, the bioluminescence was significantly stronger on day 31 after transplantation (P = 0.009). The differences between the two groups were constantly insignificant after analysis. These results indicate that both fresh and frozen–thawed testicular tissues can survive for at least 31 days after transplantation. Moreover, the vitrification group showed BLI signals comparable with those of fresh tissues. Compared with the control group, expression of the caspase-3 gene was significantly increased after vitrification (P = 0.04). Histology and IHC showed that both tissue structure and protein expression were intact in both groups.Conclusions: Transplanted vitrified ITT grafts could survive till adulthood with BLI intensity comparable to that of the fresh control. Intact cells and structures for spermatogenesis in vitrified ITT grafts were as well-preserved as those in the control group. This translational model of self-repairing vitrified ITT grafts in vivo, lends weight to the role of vitrification in prepubertal male fertility preservation.


Background
According to cancer statistics in 2020, the incidence of childhood and adolescent cancer has increased slightly, while overall mortality has been greatly reduced, declining by 68% in children and 63% in adolescents [1]. As many cancer therapies are considered gonadotoxic, fertility preservation has become an important issue for these patients [2].
According to the guidelines published by the American Society of Clinical Oncology in 2018, for prepubertal children, the only fertility preservation options are ovarian and testicular cryopreservation using methods that are currently investigational [3]. The rst and only live birth after autograft of ovarian tissue cryopreserved during childhood was reported in 2015 [4,5]. For males, previous studies in rhesus monkeys demonstrated that autologous grafting of cryopreserved prepubertal testis could produce sperm and healthy offspring [6]. However, to our knowledge, there has been no live birth following cryopreserved immature testicular tissue (ITT) transplantation in humans.
The optimal method for ITT cryopreservation remains unknown and there is no standardized protocol [2]. There are two methods for ITT cryopreservation: slow freezing and vitri cation. Currently, controlled slow freezing (CSF) is the most commonly used freezing protocol globally [7]. Vitri cation is a faster and cheaper alternative and it may avoid ice crystal formation and the ensuing injury to the tissues [8]. Since 2010, many studies have compared the feasibility of these two methods in both animal and human models, and the results are con icting. Most of the studies evaluated the tissue by in vitro morphology and immunohistochemistry (IHC) assessment instead of the function of the tissue transplanted in vivo.
Two studies comparing CSF and vitri cation for human ITT cryopreservation have demonstrated that vitri cation is able to maintain the proliferation capacity of spermatogonial cells and may be a feasible alternative [8,9]. However, more data are necessary before vitri cation could be widely used in the real world.
The development of noninvasive bioluminescence imaging (BLI) makes it possible to follow tissuespeci c luciferase expression in transgenic mice and monitor the biological processes such as signaling or protein interactions of transplanted tissues in vivo [10]. As described in our previous study, we used FVB/N-Tg (PolII-luc) Ltc transgenic mice and showed that BLI is a viable tool for assessing the e cacy of germ cell transplantation in vivo in a transgenic male mouse model [11]. Our team has used this technology to track the survival of mouse ovarian iso-and allografts [12], the effects of immunosuppressant treatment after allotransplantation of ovarian grafts [13], and the fate of cryopreserved murine ovarian grafts [14]. These previous studies mainly focused on ovarian grafts since the BLI technology is rarely used in experiments for fertility preservation of ITT.
While testicular cryopreservation is the only option available for fertility preservation in prepubertal boys, this method is still considered experimental. More studies are necessary not only to optimize the protocol, but also to make this technology more user friendly. The objective of this study was to investigate the feasibility of vitri cation for ITT cryopreservation. We studied the apoptosis of thawed ITT, and the histology after transplantation for 31 days. More importantly, we used BLI to monitor the in vivo fate of cryopreserved testicular grafts longitudinally to determine their biological activity in real time.

Mice
The donors were 3-week-old immature male FVB/N-Tg (PolII-luc) Ltc transgenic mice with an H-2 haplotype (H 2 q ). These mice were created by the transgenic service of Level Biotechnology (New Taipei City, Taiwan), and the generation process was described in our previous studies [11,13,14]. In brief, after pronuclear microinjection of the PolII-Luc transgene into the FVB/N embryos, they could encode a 712-bp mouse RNA polymerase II promoter (PolII) and a modi ed re y luciferase cDNA (Promega pGL-2). The animals were hemizygotes, and could express the transgene for luciferase (luc) and transmit this gene to their offspring. The recipients were 3-week-old immature FVB/NJNarl wild-type male mice with an H2q.
We obtained these mice from the National Laboratory Animal Center (Taipei City, Taiwan).
A total of 12 pairs of donor and recipient mice were included in our experiments. They were all bred in the animal house of Taipei Medical University at a temperature of 22-24 °C and 12/12 h light/dark regimen.
All procedures were reviewed and approved by the Animal Experimental Committee at the Taipei Medical University, in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

Experimental design
The study was divided into three parts ( Figure 1). First, we removed the 3-week-old donors' gonads, and a total of 24 donor mice were divided into two groups. In the vitri cation group, the testis from the donor mice were vitri ed and thawed before transplantation. For the other 12 mice in the control group, the ITT was transplanted to the recipients immediately. After thawing, we measured the level of expression of caspase-3 by real-time reverse transcription polymerase chain reaction (RT-PCR). Each transplanted testicular graft was measured volume 10 µg. After washing in Dulbecco's phosphate buffered saline (DPBS; Gibco ® ; Thermo Fisher Scienti c, USA), the grafts were implanted in the scrotum of each recipient.
We monitored all the transplanted grafts in vivo using BLI technology for 31 days until adulthood. On day 31, we removed the grafts and evaluated the tissue by hematoxylin and eosin (H&E) staining and IHC.

Preparation of donor ITT
The immature male FVB/N-Tg (PolII-luc) Ltc transgenic donor mice were euthanized by cervical dislocation under iso urane anesthesia. Their testes was isolated, and the tunica albuginea was immediately removed into medium and maintained at 4 °C. The seminiferous tubules were isolated and cut into pieces less than 0.5 mm. Tissues were transferred to a 1.5 mL microfuge tube and washed three times with DPBS before cryopreservation or transplantation.

Cryopreservation protocol
The vitri cation protocol was based on previous studies [15][16][17]. The vitri cation solution used consisted of 1.5 M dimethyl sulfoxide (DMSO, D2650, Sigma, USA), 0.1 mol/L sucrose (S1888, Sigma, USA), 10% fetal bovine serum (Biological Industries, Israel), 1% penicillin streptomycin in L-15 medium (20183-027, Gibco ® ; Thermo Fisher Scienti c). To achieve equilibration, 1 mL of cryoprotectant was gently introduced every minute with orbital shaking for 10 min. The tissues were incubated for 5 min at room temperature until precipitation of the ITT. The tissues were divided into 30 mL for each centrifuge and transferred to a foil (CoolRack ® ; Corning Inc., New York, USA) at 4 °C. Finally, the foil was immersed in liquid nitrogen and the ITT was transferred to a cryovial (Biomate, Taiwan) for 1 month. The warming procedure involved removing the ITT from the liquid nitrogen and quickly plunging it into a prewarmed 37 °C solution containing sucrose (1 M). The ITT was placed on an orbital shaker and DPBS was introduced at the rate of 1 mL per minute to lower the concentration of DMSO to 0.15 M. Before further assessment, the ITT was washed twice with DPBS.

Assessment of thawed ITT recovery
After general anesthesia with Zoletil (20-40 mg/kg, Virbac) and Xylazine (5-10 mg/kg, Bayer), we proceeded to the transplantation procedure and the skin was shaved around the testicular region. We performed unilateral orchiectomy and re lled the space with 10 mL of fresh or thawed seminiferous tubules. Finally, we closed the wound and a dose of Carprofen (5 mg/kg, Selleck Chemicals, USA) was given.

Real-time polymerase chain reaction
Total RNA was extracted with the RNA extraction kit (Qiagen, USA) and synthesis of the cDNA was done with the SuperScript III synthesis kit (Invitrogen, USA Transplantation and in vivo BLI imaging assessment Bioluminescence imaging was obtained using the In Vivo Imaging System 200 (Xenogen Corp., USA). The recipients were injected with D-luciferin intraperitoneally (150 mg/kg, BSAL-8220; Biosynth Carbosynth ® , USA) 10 min before imaging, anesthetized (1%-3% iso urane, Abbott, USA), and placed into a light-tight camera box on the stage of the imaging chamber. An overlay image (black-and-white picture) was taken with the aid of a light inside the imaging chamber. Luminescence was quanti ed using Living Image software. Luminescence was quanti ed by summing pixel intensities within the region of interest, as described [10].

H&E staining
For H&E staining, the testes of mice were xed in Bouin's solution (HT10132, Sigma-Aldrich). Then, the tissues were embedded in para n and the sections were cut at 5 mm. Histological and morphological changes of the testicular structures were examined under light microscopy [19].

Statistical analysis
We performed all the statistical analysis using Statistical Package for the Social Sciences version 25.0 (SPSS; IBM, USA). The changes of the photons after transplantation were measured by the following equation: (measurement on a certain day -measurement at baseline) / measurement at baseline. Differences between the vitri cation group and control group were compared using Student's t-test, and a P value < 0.05 was considered statistically signi cant.

Effect of the vitri cation intervention on caspase-3 mRNA expression within ITT
We detected the expression of the apoptosis-related caspase-3 gene in ITT immediately after vitri cation [20]. As compared with the control group, the expression of this gene was signi cantly increased after vitri cation (P = 0.04) (Figure 2).
In vivo BLI of mouse testicular grafts in fresh and frozenthawed testicular transplant groups on days 1, 2, 5, 7, and 31 after transplantation After we removed one of the recipients' testes, we replaced it with fresh or frozen-thawed ITT from the FVB/N-Tg (PolII-luc) Ltc transgenic mice. We traced the survival of the graft by in vivo BLI on days 1, 2, 5, 7, and 31 after transplantation (Figure 2A). For both the vitri cation and the control groups, the bioluminescence decreased between day 2 and day 5. After day 5, the bioluminescence showed an upward trend until day 31. Compared with day 1, the bioluminescence was signi cantly stronger at day 31 after transplantation (9.3 ´ 105 photons/s versus 15.4 ´ 105 photons/s, P = 0.009). The bioluminescence of the vitri cation group was initially stronger than that of the control group. However, the signals were lower than the control group after day 7 ( Figure 2B). The differences between the two groups failed to reach signi cance ( Figure 2C). These results indicate that both fresh and frozen-thawed testicular tissues can survive for at least 31 days after transplantation. Moreover, the vitri cation group revealed bioluminescence signal intensity that was comparable with that of the fresh tissues.
Histology and IHC staining analysis of ITT grafts on day 31 after transplantation Since the BLI signals revealed that both fresh and frozen-thawed testicular tissues could survive for at least 31 days after transplantation, we determined their structure and function by histology and IHC, 31 days after transplantation. Seminiferous tubules could be observed in both groups, but the structure was more intact in the control group ( Figure 3A).
Proliferating cell nuclear antigen (PCNA) is a useful marker for mitotically proliferating spermatogonia, but not for spermatocytes that have just entered meiosis [21]. Sperm acrosome membrane-associated protein 1 (SPACA1) is a membrane protein with a function in sperm-egg fusion [22]. The expression of luciferase indicated that the cells originated from the transgenic ITT. Sox-9, 3b-HSD, and DDX4 represent Sertoli cells, Leydig cells, and germ cells, respectively [23][24][25]. The expression of the above antigens were all detectable in both the vitri cation and control groups. We assumed that the ITT integrities were preserved during the process of vitri cation, consistent with the result of BLI imaging. However, compared with the control group, the protein expression was decreased in the vitri cation group ( Figure 3B).

Discussion
Many studies have investigated the feasibility of vitri cation for ITT cryopreservation. To the best of our knowledge, this is the rst study to report the survival of ITT in vivo by adopting the BLI imaging system.
Apoptosis of the vitri ed ITT was noted before transplantation. During longitudinal observation of the bioluminescence in vivo for 31 days, we con rmed that the tissues were viable after transplantation in both the vitri cation and the control groups with comparable bioluminescence intensity. The intensity was signi cantly stronger on day 31 compared with the rst day, indicating the possibility of tissue selfrepair after transplantation. On day 31, the histology and IHC demonstrated that the vitri ed tissues had preserved the structures and cells for spermatogenesis.
The aim of our study was to adopt the translational model of BLI signals in transgenic mice to track the survival of the grafts in vivo. In our previous study, this animal model was shown to be useful for quantifying germ cells in vitro and assessing the e cacy of germ cell transplantation in vivo [11]. In the present study, our results indicated that at follow-up on days 1, 2, 5, 7, and 31 after transplantation, bioluminescence signals were similar between the vitri cation group and the control group. This nding suggests that ITT can recover from the vitri cation-thawing process and establish revascularization in a manner similar to fresh tissues from the beginning of transplantation. This observation is comparable with earlier in vitro studies that reported similar outcomes with vitri cation after CSF or with fresh controls [9,26,27]. Unlike in ITT, ovarian cryopreservation by slow freezing may compromise ovarian reserve through cryoinjury and ischemia. We found the BLI signals persisted lower in the slow freezing group on days 1, 3, 5, 7, and 10 than the fresh controls after transplantation [14]. The process of revascularization takes 2 to 7 days to complete, depending on the size of the implant [28]. The bioluminescence intensity decreased between day 2 and day 5, and then increased linearly. The BLI signals were signi cantly higher on day 31 than on day 1, indicating the presence of revascularization after day 5, and the subsequent restoration of function of the ITT grafts.
According to previous reviews, eight studies have compared vitri cation to slow freezing for ITT cryostorage [2,7]. In 2010, Abrishami et al. reported that ITT vitri cation could maintain cell viability and restore spermatogenesis after xenograft [29]. They found that exposure to DMSO for 5 min yielded numerically higher cell numbers than in grafts exposed for 15 or 30 min. Accordingly, we limited DMSO exposure of our grafts to less than 10 min. Curaba et al. compared vitri cation and slow freezing in terms of IHC and tissue integrity. They con rmed that vitri cation was a promising approach, but additional studies should be conducted in vivo to assess completion of spermatogenesis [16]. The same authors also published a case report assessing vitri cation of human ITT, and revealed that the histology characteristics of spermatogonia and Sertoli cells were preserved [8]. Gouk et al. focused on harvesting spermatogonial stem cells (SCCs) for fertility preservation. They found that vitri cation maintained postwarming cell viability and function signi cantly better than conventional slow and rapid freezing protocols [15]. Baert et al. and Poels et al. both agreed that vitri cation is an effective strategy to maintain the proliferative capacity of SCCs and integrity of ITT [9,17]. Poels et al. was the rst group to adopt a human ITT xenotransplantation model. They observed spermatogonia proliferation 6 months after transplantation, but then there was a blockage at the pachytene stage. Time-consuming CSF protocols are commonly used in human testicular tissue banking. Baert et al. have investigated the alternatives to conventional CSF using testicular tissues from 14 adult patients. In the vitri cation group, they found increased numbers of seminiferous tubules displaying a ruptured epithelium and considered this method to have a negative impact on spermatogonial number [30]. On the other hand, Dumont et al. reported the superiority of the vitri cation protocol in terms of testicular structure maintenance, tubular morphology, and tissue function [31]. In a recent review, these authors concluded that the results of comparison between vitri cation and slow freezing for ITT were still con icting, and most of them were limited to in vitro animal studies [2].
In vitro cell culture is an interesting method to avoid re-introduction of malignant cells to cancer patients, especially with hematological malignancies [32]. Sato and Ogawa were the rst to show complete spermatogenesis from cryopreserved ITT after it was thawed and cultured in vitro [33]. Furthermore, Yokonishi et al. suggested that tissues undergoing vitri cation showed spermatogenesis similar to that of unfrozen control tissues [26]. The cryopreservation method is not as critical as culture conditions, such as type of culture medium or temperature. Dumont et al. also con rmed that in vitro spermatogenesis maturation strongly modi ed apoptosis and autophagy-related protein levels, and the impact of cryopreservation was minimal at the end of the culture [27].
The limitation of this study is that the BLI signals could represent tissue viability only, and not its ability for spermatogenesis. Despite the fact that the vitri cation group revealed substantial BLI signals similar to the control group, we could not detect spermatids during the morphology evaluation on day 31 after transplantation in either group. Histology and IHC con rmed the presence of some important cells and structures for spermatogenesis. Others have reported live spermatogenic cells recovered at 2 months after transplantation and produced the rst live birth of rabbit offspring [34]. Recently, the same group retrieved post-meiotic spermatids 8 to 12 months after transplantation, and successfully produced offspring from autologous grafting of cryopreserved prepubertal rhesus monkeys [6]. The lack of spermatogenesis in our study may simply re ect insu cient observation time.

Conclusion
Apoptosis was present in vitri ed ITT before transplantation. Transplanted vitri ed ITT grafts could survive until adulthood with comparable BLI intensity to the fresh control while stained vitri ed ITT grafts indicated intact cells and structures preserved for spermatogenesis. This translational model of selfrepairing vitri ed ITT grafts in vivo lends weight to a role for vitri cation in prepubertal male fertility preservation.

Declarations
Ethics approval and consent to participate  Figure 1 Study design owchart. The donor mice were 3-week-old immature male FVB/N-Tg (PolII-luc) Ltc transgenic mice with an H-2 haplotype (H 2 q ). Their immature testicular tissues (ITT) were removed and fragmented. Then, the ITT were either vitri ed and thawed before transplantation or transplanted directly to wild-type recipient mice. After anesthesia and D-luciferin injection, we used the In Vivo Imaging System 200 (IVIS 200) to monitor the bioluminescence (BLI) signal intensity in vivo. Finally, the quanti ed BLI signal data were exported to the computer for further longitudinal research.

Figure 2
Caspase-3 mRNA expression was determined by real-time qPCR. Its expression was signi cantly higher in the vitri cation group (P = 0.04). were all not signi cant between the two groups.