Use of In Ovo Chorioallantoic Membrane Engraftment to Culture Testes from Neonatal Mice

Abstract

Many attempts have been made to culture germ cells in vitro by mimicking their development in vivo. The objective of this study was to establish an alternative method of xenotransplantation by developing a new approach for the rapid induction of spermatogenesis by using the chorioallantoic membrane of developing chicken embryos. Fertilized chicken eggs were incubated for 7 d, after which a small window was cut into the shell of the egg. We then transplanted testes from 7- to 8-d-old B6D2F1 mice onto the vessels of the chorioallantoic membrane and incubated them at 35.0 °C for 14 d or 37.5 °C for 12 d. After this in ovo CAM (iCAM) culture, the survival rates of the eggs and testes were assessed histologically and immunohistologically. The transplanted testes in the chicken embryos that survived were supported by the CAM, with an associated chronic vascularization response. The testes cultured at 35.0 °C had lower rates of generation and higher rates of death than did those cultured at 37.5 °C. Histologic examination of the testes cultured at 37.5 °C revealed the presence of spermatogonia and primary spermatocyte-like germ cells in the seminiferous tubules. The number of cells positive for synaptonemal complex protein 3 in the seminiferous tubules was significantly higher than that in the noniCAM-cultured testes from control mice. These results suggest that iCAM culturing of neonatal donor testis induces androcyte development. This method could be the foundation for a method that would enable in vitro spermatogenesis.

In recent studies of in vitro spermatogenesis, many attempts have been made to culture germ cells in vitro by mimicking their development in vivo. However, the complexity of germ cell development has hampered the induction of immature germ cells into functional gametes under culture conditions. It is well known that spermatogenesis is one of the longest processes of sequential cell proliferation and differentiation, taking more than a month for progression from spermatogonial stem cells, through meiosis, to sperm formation. Male germline cells in mice can only be cultured from pachytene spermatocytes to round spermatids to produce the next generation.¹² Until recently, researchers succeeded only in inducing the early steps of spermatogenesis in vitro.¹⁶

In the first study to overcome this difficulty, neonatal mice testis, containing only gonocytes or primitive spermatogonia as germ cells, produced spermatids and sperm in vitro after tissue culture for 2 mo.¹⁵ The embryos derived from these cultured spermatozoa yielded live and normal offspring. In the approach, the culture system widely preserved the cytoarchitecture of the gonad, many endogenous factors were produced and released by the mostly intact seminiferous epithelium, and associated somatic cells regulated the germ cells in a manner similar to the in vivo condition.¹⁵ Other investigators showed that in dissected tissue, the supply of oxygen and nutrients is disturbed and that the spermatogenic process consequently is often disrupted and continues, at best, at low efficiency.¹ Therefore, as a valuable alternative approach, a culture system should be established for the development of spermatogenesis in vitro.

The avian chorioallantoic membrane (CAM) is the outermost extraembryonic membrane that lines the noncellular eggshell membrane. The CAM is formed by the fusion of the splanchnic mesoderm of the allantois to the somatic mesoderm of the chorion. CAM vessels grow rapidly until day 11, and the vascular system attains its complete structure on day 18 of incubation, just before hatching.¹³ The CAM serves as a support for the extraembryonic respiratory capillaries and actively transports sodium and chloride from the allantois sac and calcium from the eggshell into the embryonic vasculature.¹⁷ The CAM plays a role in the neovascularization of developing tissues and provides an immunologically protected site in which it is possible to observe the development of blood vessels in the living egg. The extraembryonic vessel system of CAM is naturally immunodeficient.⁹ In addition, the degree of neovascularization of the donor testis by host blood vessels can be determined histologically by the presence of nucleated avian erythrocytes in blood vessels.⁵ For these reasons, the CAM system has been applied to implantation studies with tumors, adipocytes, and cartilages as well as ovarian tissues, but no approach to in ovo spermatogenesis has been reported previously.^4,6-8,10,18 Our current study aimed to establish a novel in ovo system for culturing the testes of mice on the CAM and to investigate whether in ovo CAM (iCAM) culture supports the induction of spermatogenesis and meiosis of the cultured germ cells.

Materials and Methods

Principles of laboratory animal care were followed during this study, and all procedures were conducted in accordance with guidelines of the Ethics Committee for Care and Use of Laboratory Animals for Research (Niigata University, Japan). B6D2F1 mice were used throughout the experiments. Mice were kept in polycarbonate cages (25 × 40 × 20 cm) under controlled conditions with lights on at 0600 and off at 1800. They were given food and tap water in libitum.

Testes collection and CAM culture.

B6D2F1 mice (age, 7 to 8 d) were euthanized by cervical dislocation, and both testes were excised. Fertilized Julia chicken eggs were obtained from I Hiyoko (Sanjyo, Niigata, Japan). The eggs first were incubated in an incubator (BH-03, Showa Furanki, Tokyo, Japan) for 7 d at 37.5 °C under 52% humidity. Each egg then was washed with 70% ethanol, after which a triangular window was cut into the pointed pole of the shell by using a dental drill, according to a modified, previously described procedure.^9,13 This window was enlarged to approximately 10 × 10 mm to reveal the underlying embryo and CAM vessels (Figure 1 A). The testis immediately was transplanted onto the CAM. Thereafter, the shell window was sealed, and the egg was returned to the incubator. The survival rates of the transplanted testis and the host egg were evaluated after either 12 d of iCAM culture at 37.5 °C or 14 d at 35.0 °C.

Figure 1.

In ovo chorioallantoic membrane (iCAM) culture of mice testes. (A) Day 7 chick embryo. (B) Testicular transplant on the CAM on day 19. Scale bar, 5 mm.

Histologic analysis.

After iCAM culture, the mouse testes were removed from the eggs and fixed in Bouin fluid for more than 72 h. They then were processed through graded ethanol for dehydration and embedded in paraffin wax. Serial sections were cut every 5 μm and then deparaffinated. Subsequently, the sections were stained with hematoxylin and eosin for microscopic evaluation of the testicular tissue and differentiation of the male germ cells.¹⁰ We examined the diameter of the seminiferous tubules and the total number of germ cells in random fields of 10 seminiferous tubules. Only seminiferous tubules that had completely round tubules were selected for analysis, and germ cells were classified based on their stage of development, from spermatogonia to spermatocyte. Testes from control B6D2F1 mice were collected and evaluated at the ages of 7 to 8 (same time point as at the beginning of iCAM culture) and 19 to 21 (same time point as at the end of iCAM culture).

Immunohistologic analysis.

Immunohistochemistry was performed to detect meiotic cells by screening for synaptonemal complex protein 3 (SCP3; Abcam, Cambridge, UK). SCP3 is a meiosis-specific proteinaceous structure that is required for the pairing and segregation of homologous chromosomes at the first meiotic division.² Therefore, it is considered a marker of meiosis.

Serially cut deparaffinized sections of the iCAM-cultured testes were washed with PBS and then treated with 0.3% H₂O₂ in methanol for 30 min at room temperature, to eliminate endogenous horseradish peroxidase. After the sections were washed again with PBS, they were blocked with PBS containing 1% normal goat serum for 30 min at room temperature. Subsequently, SCP3 antibody diluted in PBS (1:2000) was dropped on the sections, and they were incubated at 4 °C overnight. The next day, the sections were washed with PBS and then reacted against biotinylated goat antirabbit IgG antibody (1:200). The sections were stained for 30 min by using the Vectastain ABC Elite Kit (Vector Laboratories, Burlingame, CA). After the sections were washed with PBS, they were stained with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories) and evaluated.

Immunofluorescent assay.

Deparaffinized and rehydrated sections were steeped in 10 mM citric acid containing 0.05% Tween 20 and then placed in an autoclave for heat-mediated antigen retrieval. The sections were blocked with PBS containing 5% normal goat serum for 20 min at room temperature. Subsequently, SCP3 antibody diluted in PBS was dropped on the sections, which then were incubated at 4 °C overnight. On the following day, the sections were washed with PBS and reacted for 1 h with dilute goat antirabbit IgG conjugated with Alexa 488 (1:100). After the sections were washed with PBS, they were observed by fluorescence microscopy (BX60, Olympus, Tokyo, Japan).

Statistical analysis.

To determine the association between 2 variables in Table 1, a χ² test was applied. The 2-tailed P values less than 0.0001 to determine significant differences between variables was calculated by using Quickcels software (version 2013, GraphPad Software, San Diego, CA). The data in Table 2 underwent ANOVA and the Fisher protected least significant difference post hoc test (StatView, Abacus Concepts, Berkeley, CA). Data are expressed as the mean ± SEM; P values of less than 0.05 indicated statistical significance.

Table 1.

Vascularization (%) of the testicular transplants and viability of the host embryos

Treatments	Vascularization of live testicular transplants	Live host embryos	Dead host embryos
35.0 °C for 14 d (n = 38)	11 ± 29a	14 ± 37a	24 ± 63a
37.5 °C for 12 d (n = 37)	29 ± 78b	34 ± 92b	3 ± 8b

Different superscripts within the same column denote significant differences (P < 0.0001).

Table 2.

Diameter of seminiferous tubules and number of germ cells in random seminiferous tubules in uncultured controls and after iCAM culture at 37.5 °C

	Day 7–8 (n = 5)	Day 19 (n = 5)	37.5 °C (n = 5)
Diameter (μm) of seminiferous tubules	64.9 ± 1.2a	134.0 ± 2.8b	75.4 ± 3.4c
No. of germ cells per seminiferous tubule	34.8 ± 0.9a	110.6 ± 3.9b	55.3 ± 2.8c

Values are represented as the mean ± SEM. Different superscripts within the same row denote significant (P < 0.05) differences.

Results

Testis tissue observation.

The 7- to 8-d-old mice testes that survived transplantation and incubation in the egg were supported by the CAM and had visible blood vasculature after iCAM culture. The mouse testis appeared light pink before it was transplanted onto the CAM, whereas successfully transplanted testes cultured both at 35.0 °C and 37.5 °C were deep red (Figure 1 B). In contrast, testes that had failed to survive transplantation and culture were light pink, as was pretransplantation testis, or cream.

The vascularization of the transplants and viability of host embryos are reported in Table 1. Testes cultured at 35.0 °C showed lower rates of generation and higher rates of death than did those cultured at 37.5 °C. That is, the success rate after culture at 35.0 °C was lower than that at 37.5 °C.

Histologic analysis.

In iCAM cultures at both 35.0 °C for 14 d and 37.5 °C for 12 d, incorporation of the testes into the CAM occurred with an associated chronic vascularization response. The histology after testes retrieval is shown in Figure 2. The details of the iCAM-induced spermatogenesis were examined by comparison with the seminiferous tubule size and the number of germ cells generated from normal spermatogenesis in the testes of control mice (Table 2). In testes removed at 7 to 8 d after birth, the diameter (mean ± SEM) of seminiferous tubules was 64.9 ± 1.2 μm, and most of the germ cells were spermatogonial during this period (Figure 2 A). In testes removed at 19 d after birth, the seminiferous tubules were larger (134.0 ± 2.8 μm) than those obtained at 7 to 8 d, and both spermatogonia and primary spermatocytes were present (Figure 2 B).

Figure 2.

Photomicrographs sections of mice testes. (A) Normal spermatogenesis in day 7 mouse (scale bar, 10 µm). (B) Normal spermatogenesis in day 19 mouse (scale bar, 10 µm). (C) iCAM-cultured testis at 35.0 °C for 14 d. (D) iCAM-cultured testis at 37.5 °C for 12 d (scale bar, 10 µm). (E) iCAM-cultured testis at 37.5 °C for 12 d (scale bar, 10 µm). Hematoxylin and eosin stain.

Testes cultured in iCAM at 37.5 °C for 12 d had visible seminiferous tubules (Figure 2 D and E) with an average diameter of 75.4 ± 3.4 μm, which was significantly larger than that of seminiferous tubules of the day 7 control (64.9 ± 1.2 μm). In addition, the number of germ cells the testes iCAM-cultured at 37.5 °C was greater than that of the day 7 control. In testes iCAM-cultured at 35.0 °C, the seminiferous tubules were broken and contained a mix of erythrocytes and androcytes (Figure 2 C). By contrast, the control testes at 19 d after birth were similar to the iCAM-cultured testes at 37.5 °C.

Immunohistologic and immunofluorescence analysis.

In testes removed at 8 and 19 d after birth (control) and in testes cultured on CAM at 37.5 °C for 12 d, SCP3-positive cells were present in all developed seminiferous tubules. Testes removed at 8 and 19 d after birth displayed 2 types of seminiferous tubules, which either contained or did not contain SCP3-positive cells (Figures 3 and 4). The SCP3-positive cells were scattered throughout the seminiferous tubules of iCAM-cultured testis (Figure 4). However, the number of SCP3-positive cells per iCAM-cultured seminiferous tubule was greater than those for all control seminiferous tubules (Figures 3 through 5).

Figure 3.

Distributions of SCP3 in normal and iCAM-cultured mice testes. Serial sections in panels A through C and D through F (high magnification) were stained for SCP3 and visualized with 3,3′-diaminobenzidine tetrahydrochloride (brown). (A and D) Testis sections from iCAM, cultured at 37.5 °C for 12 d. (B) and (E) Testis sections from day 8 mice. (C) and (F) Testis sections from day 19 mice. Magnification, 100× (panels A through C); 400× (scale bar, 10 µm; panels D through F).

Figure 4.

Distributions of SCP3 in normal and iCAM-cultured mice testes. Serial sections in panels A through C and D through F (high magnification) were stained for SCP3 and visualized with Alexa-488 (green). (A and D) Testis sections from iCAM, cultured at 37.5 °C for 12 d. (B and E) Testis sections from day 8 mice. (C and F) Testis sections from day 19 mice. Magnification, 200× (panels A through C); 400× (scale bar, 10 µm; panels D through F).

Figure 5.

Number (mean ± SEM) of SCP3-positive cells per seminiferous tubules after iCAM culture of mice testes at 37.5 °C for 12 d. Different superscripts denote significant (P < 0.05) differences.

Discussion

Several researchers have attempted to transplant tissue to the CAM of chicken embryos to study the development of various organs and structures. Tissue placed on the CAM is vascularized rapidly, and the lack of an immune system at this stage of the chick development prevents transplant rejection. Therefore, we transplanted testes from neonatal mice onto the CAM of 7-d-old chick embryos, to test the hypothesis that culture in ovo would support spermatogenesis for use as a tool in the study of male germ cell development. First, we assessed iCAM culture conditions. The 2 temperatures that we tested (35.0 °C and 37.5 °C) were chosen for its similarity to the mouse scrotal environment and as a suitable temperature for artificial incubation, respectively. The 12-d incubation at 37.5 °C reflected the fact that eggs hatch within 21 d at 37.5 °C, whereas the 14-d duration at 35.0 °C accounts for the additional 1-d delay in hatching for each 1°C decrease in incubation temperature.

Testes cultured at 35.0 °C showed damaged seminiferous tubules and a mixture of androcytes and erythrocytes. Moreover, the rate of chick embryo death was increased at 35.0 °C, because this temperature was very low as compared with the optimal temperature (37.5 °C) for egg incubation. In contrast, after iCAM culture of testes at 37.5 °C, the membranes of seminiferous tubules remained intact. In addition, the size of seminiferous tubules and number of germ cells were significantly greater in the testes iCAM-cultured for 12 d at 37.5 °C compared with those of the day 7 control group. This finding suggests that the testicular tissue had successfully adhered to the chick embryo CAM and that angiogenesis had developed. From these results, we conclude that the optimal incubation temperature for iCAM culture is 37.5 °C.

The first wave of spermatogenesis in mice is initiated only a few days after birth and proceeds in a synchronized manner.¹¹ Key time points for appearance of particular germ cell types are well defined during this first cycle of spermatogenesis. In 7-d-old mice, the testes contain only Sertoli cells and spermatogonia in the seminiferous tubules. By postnatal day 9, early spermatocytes appear, the pachytene stage of the first meiotic prophase is initiated at day14, and late pachytene and diplotene spermatocytes are present at PND 18. Due to meiotic divisions, round spermatids appear around day 20. It is well known that mice demonstrate 12 stages in the production of mature spermatozoa, with each stage corresponding to specific segments in the seminiferous epithelium. The complete series of stages of the seminiferous epithelium constitutes a cycle, which in mice requires 8.45 d, with 33 d needed for complete spermatogenesis.³ SCP3 is a synaptonemal complex protein from paired homologous centromeres and has been implicated in the diplotene–metaphase I transition. However, other investigators showed that SCP3 was present at early leptotene and persisted through the stages of microtubule attachment to kinetochores and meiosis separation of the homologous partners.²

Second, we examined whether germ cells in the seminiferous tubules of cultured testes underwent meiosis. Immunohistologic staining using SCP3 antibody revealed SCP3-positive cells along the basilar membrane. Because mouse spermatocytes undergo apoptosis spontaneously, long-term maintenance of cultures of mice testes with intact seminiferous tubules would be difficult. However, the seminiferous tubules of iCAM-cultured testes at 37.5 °C for 12 d had significantly more SCP3-positive cells than did noncultured control tubules. This finding suggests that apoptosis did not occur in the iCAM cultures, that androcyte development had been induced, or that activation of meiosis occurred in the CAM transplants.

Vascularization is a very important factor for successful testes graft transplantation, as it is required for the rapid establishment of the blood supply that is crucial for the survival of germ cells. Spermatogenesis leading to the generation of spermatozoa is a prolonged process, requiring 35 d in mice, 61 d in bovines, and 74 d in humans.¹⁴ One report indicates that spermatogenesis with the formation of sperm with flagella took 25 to 60 d in vitro.¹⁵ From the results of our study, we propose that obtaining viable sperm in vitro requires an environment capable of maintaining long-term cultures. The iCAM culture system is limited by the incubation period of the host embryo, which is 21 d. Tissue transplants can be maintained for only 12 to 14 d after they are placed on the CAM. This duration may not be sufficient to determine whether activation of meiosis occurs in the CAM transplants. However, our results demonstrate the presence of spermatogonia and primary spermatocyte-like germ cells in the seminiferous tubules of testes iCAM-cultured at 37.5 °C. The number of SCP3-positive cells per seminiferous tubule was significantly increased in iCAM-cultured tubules compared with tubules collected from control mice on days 8 and 19. Therefore the germ cells in the testes grafts can be considered to have entered the leptoten stage of the meiosis, as indicated by the presence of SCP3-positive cells. These findings suggest that androcyte development was induced in the iCAM cultures of neonatal donor testis and that a specific factor in the blood of chicken embryos may induce spermatogenesis, because we noted nucleated erythrocytes (derived from the chick embryos) in the donor seminiferous tubules under iCAM culture at 37.5 °C. Perhaps a serial iCAM culture system can be established to accommodate the prolonged time needed to mature spermatozoa.

In conclusion, we have shown that, by using a simple and practical method, neonatal mice testes can be maintained through an iCAM culture system to yield germ cells in the leptoten stage of meiosis. The iCAM culture model we used in the current study provides not only a novel tool for the induction of spermatogenesis in ovo but also an in vitro means to study the mechanism of spermatogenesis. In the future, this system may become a valuable alternative method for testicular xenotransplantation and the study of spermatogenesis.