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Development-promoting effect of chicken embryo membrane on chicken ovarian cortical pieces of different age
PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Development-promoting effect of chicken embryo membrane on chicken ovarian cortical pieces of different age J. H. Yuan,* J. S. Gao,* Z. F. Zhan,† H. W. Liu,* W. J. Jin,* and Z. D. Li*1 *Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricutural University, Beijing, China, 100193; and †National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China, 100193 ABSTRACT Ovarian cortical tissues of various ages of chicken were grafted underneath chorioallantoic membrane (CAM) of 6-d-old chicken embryo and the grafts were collected on d 0, 2, 4, 8, or 10. The tissue sections were prepared to examine the development of follicles in grafts and the proliferative ability of follicles was examined by immunohistochemistry analysis of proliferating cell nuclear antigen. The results indicated that the development of follicles in cultured chicken ovarian cortical pieces progressed smoothly and slowly similar to those in vivo, and the environment of chicken embryo was more suitable for the synchronous development of oocyte and follicular cells in the larger follicles. Meanwhile, the synchronous development could
be more easily achieved in the chicken ovarian tissues of 12- to 15-wk chicken. Neither oocyte nor granulosa cells but the thecal cells of follicles developed in grafts of 13- to 15-d chicken within 10 d of culturing in ovo, and the CAM can ease follicular atresia in chicken ovarian grafts of 12- to 15-wk chicken. The proliferative performance of follicles in the grafts was not influenced by the environment of chicken embryo. These results indicated that the chicken embryo has the ability to support slow development of primordial and primary follicles in grafted ovarian cortical tissues of chickens of different ages. The CAM system may also prove useful for the study of early follicle development with further information.
Key words: chicken, follicle development, in ovo, chicken embryo 2009 Poultry Science 88:2415–2421 doi:10.3382/ps.2008-00555
INTRODUCTION The domestic fowl provides a unique model for the study of the mechanisms involved in follicular development. The single left ovary contains follicles of various sizes and developmental stages. As in mammals, all ova within the chicken ovary are primary oocytes until shortly before ovulation (Hodges, 1974). Domestic hen ovarian follicles are organized during development, primary oocytes enclosed by the vitelline membrane become organized into a primordial follicle (up to ~80-μm diameter) after the recruitment of presumptive granulosa cells, and the perivitelline membrane is subsequently formed by granulosa cells. The initiation of primordial follicle growth to the primary follicle stage is associated with the formation of the theca layer (Johnson, 1999a,b). The constant viability and development of ovarian follicles is intricately controlled by endocrine (e.g., gonadotropins), paracrine, and autocrine factors, ©2009 Poultry Science Association Inc. Received December 20, 2008. Accepted May 16, 2009. 1 Corresponding author: [email protected]
plus neurochemical and neurohumoral factors (Johnson, 2000). Moreover, a great deal of environmental factors, such as nutrition, can influence follicular development and oocyte quality, and hence fertility (Garnsworthy and Webb, 1999; Webb et al., 1999). The activation and subsequent growth of primordial follicles still remain poorly understood. Considerable progress has been made in the culture of mammal ovarian tissue such as mouse (eppig and O’Brien, 1996; Webb et al., 2004; Gigli et al., 2005), bovine (Wandji et al., 1996; Braw-Tal and Yossefi, 1997), and human (Roig et al., 2006). In regards to reproduction of poultry, chimeras had been obtained from injection of frozen-thawed blastodermal cells (Kino et al., 1997), and offspring have been produced from orthotopic transplantation of chicken ovaries (Song and Silversides, 2007a) and cryopreserved chicken testicular tissue (Song and Silversides, 2007b). Follicles isolated from the ovary of hens have been cultured in vitro more than 5 d (Du et al., 2006). However, the manipulation was limited to the embryo and hatching chicken gonads, or the follicles were detached from the ovarian tissues. Development of a culture system for ovarian cortical tissues will contribute to our understanding for
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regulating mechanisms of follicular survival, growth, and differentiation, which are also prerequisites for the protection and use of avian reproduction resources. Grafting tissue to the chorioallantoic membrane (CAM) of chick embryos to study the development of various organs and structures is a technique that has been used by developmental biologists for decades (Rudnick, 1944; Nakada et al., 1998; Kunzi-Rapp et al., 1999; Katoh et al., 2001; Leng et al., 2004). The embryonic CAM is highly vascularized and the lack of a functional immune system before d 17 of chick embryo development prevents rejection; tissue grafted to CAM is usually rapidly vascularized. When the fetal bovine ovarian cortical pieces were placed between the CAM and the yolk sac of 6-d-old chick embryos, the primordial follicles were not activated in the grafts during 10 d in ovo; however, primordial follicles retained their ability to activate (Cushman et al., 2002). It has also been indicated that all the frozen-thawed human ovarian cortical grafts showed adhesion, and most of the follicles were healthy (Martinez-Madrid et al., 2008). However, no study has analyzed the development of follicles in the grafts in detail. Meanwhile, as the same species to the chicken embryo, no work has been done to study the supporting ability of chicken embryos to the development of chicken ovarian tissues. Therefore, ovarian cortical pieces of adult and developing chicken were grafted underneath the CAM of 6-d-old chicken embryo to examine whether the CAM model could be used as an in ovo system to culture chicken ovarian cortical tissue and to find out to what extent the grafts can be developed.
licles of around 500 μm in diameter visible to the naked eye were removed with sterile scissors. Then, the ovary was washed in warmed Dulbecco’s PBS 4 times after being in 70% alcohol for 20 s and cut into small pieces of 0.5 to 1 mm3 (Supplemental Figure 1A to C; all supplemental figures can be found online at http://ps.fass. org/content/vol88/issue11/). The cortical pieces were placed between the CAM and the yolk sac (1 piece/egg) of 6-d-old chick embryos to culture as used by Cushman et al. (2002), and the tissues were retrieved on d 0 (uncultured tissues), 2, 4, 8, and 10 (Supplemental Figure 1D to F).
Histological Analysis
Female Single Comb White Leghorn chickens of various ages were raised in floor pens. The chickens had free access to food and water. The breeder company-recommended lighting regimens were maintained throughout the experiment. Five birds of each age were slaughtered by cervical dislocation to collect ovaries on d 13 to 15, wk 12 to 15, and wk 50 to 55. Hens (in 50 to 55 wk) were kept in individual cages and oviposition time was recorded at hourly intervals in the morning and at 1700 h, and those with regular laying sequences of 6 eggs or more were selected randomly. The ovarian tissues were obtained from hens 2 h after oviposition of the first egg in the sequence. All animal experimentation was conducted in accordance with the regulations of the animal ethics committee set by China Agricultural University.
Chicken ovarian cortical pieces collected were fixed in 10% neutral buffered formalin diluted with 0.02 mol/L of PBS (8.5 mol/L of NaCl, 2.8 mol/L of Na2HPO4, and 0.4 mol/L of NaH2PO4; pH 7.2 to 7.6) for 24 h. For histological analysis, tissue samples were embedded in paraffin and sections of 5 μm were cut. Briefly, more than 8 ovarian cortical tissues from each group were processed for histological examination using the standard hematoxylin and eosin method (Supplemental Figure 1 G to I). The follicles were measured in each of 2 serial sections from the largest cross-section through the center of the ovary and averaged in each category (Parrott and Skinner, 1999; Nilsson and Skinner, 2002) and only follicles containing oocytes with a visible nucleus were measured. The sections were photographed using a Leica DFC 320 digital camera (Leica Microsystems, Wetzlar, Germany) and the diameters of follicles and oocytes were measured by Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Accordingly, 2 measurements were taken when measuring diameters and the second measurement originated at a right angle from the midpoint of the first measurement. The 2 measurements were then averaged and expressed as the diameter of the structure. The follicles in chicken ovarian pieces of 12- to 15-wk and 50- to 55-wk chicken were classified into 4 groups according to diameters: <100, 100 to 190, 200 to 290, and >300 μm. Follicles with diameters less than 100 μm were classified as primordial follicles, and more than 100 μm was considered developing follicle. The follicles with one or more of the following characters were deemed atretic follicles (such as Supplemental Figure 2I and L): a shrunken and irregularly shaped oocyte, the presence of multiple large vacuoles, the presence of blood cells or cellular debris in the ooplasm, dissociation among granulosa cells, granulosa detached from basal lamina, invasion of granulosa, loss of identity of granulosa and theca interna (Gupta et al., 1988).
Culture of Ovarian Cortical Tissues In Ovo
Immunohistochemistry
Collected ovary was transferred into sterile Dulbecco’s PBS (Sigma, St. Louis, MO); for the 12- to 15-wk and 50- to 55-wk chicken, yellow follicles and white fol-
A mouse monoclonal antibody raised against human proliferating cell nuclear antigen (PCNA; Boster, Wuhan, China) was used to detect proliferating cells
MATERIALS AND METHODS Preparation of Ovarian Cortical Pieces
CHICKEN OVARIAN TISSUE GRAFTED TO CHICKEN EMBRYO MEMBRANE
in cultured and noncultured ovarian cortical tissues. Slides were deparaffinized and rehydrated. A commercial Streptavidin-Biotin-Enzyme Complex kit (BSCXBiotech, Wuhan, China; Zhan et al., 2008) was used for immunohistochemistry. Briefly, sections were incubated in 3% H2O2 for 20 min at room temperature to block endogenous peroxidase, incubated sequentially with 5% BSA for 20 min after microwave antigen recovery, PCNA monoclonal antibody diluted 1:400 in PBS at 4°C overnight, and then corresponding biotinylated secondary antibodies against mouse and Streptavidin peroxidase. Subsequently, binding of the primary antibody was detected with diaminobenzidine. Sections were counterstained with hematoxylin. In the negative control, the primary antibodies were substituted with PBS.
Statistical Analysis Statistical analyses were performed using the GLM procedures of SAS (version 8.2, SAS Institute Inc., Cary, NC). All data are expressed as the mean ± SEM. Differences between means were determined using the Duncan’s multiple range test. A P-value <0.05 was considered statistically significant.
RESULTS AND DISCUSSION Development of Grafted 50- to 55-wk and 12- to 15-wk Chicken Ovarian Cortical Tissues In Ovo Chicken embryo maintained the healthy morphology of follicles even on d 10, and blood cells could be found as early as d 2 in ovo (Supplemental Figure 2A to P). Compared with uncultured ovarian pieces, the percentage of <100-μm follicles in tissues decreased (P < 0.05) after culture in chicken of both 50 to 55 wk (Figure 1A) and 12 to 15 wk (Figure 1B), whereas >100-μm
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follicles proved the opposite. However, smaller follicles in the ovarian cortical pieces were still more than larger follicles in the number after culture as control. The average diameter of follicles in each group did not differ nearly (P > 0.05) from each other before or after culture (Figure 2A and B) despite increasing (P < 0.05) diameter of 100- to 190-μm follicles in grafts of 12- to 15-wk chicken on d 4 than 0 (Figure 2B), which indicated that the development of follicles in the grafts was moderate. But in all the follicles in the grafts of 50to 55-wk chicken (Figure 3A), and in follicles of <300 μm in 12- to 15-wk chicken (Figure 3B), the volume ratio of oocyte and follicle decreased (P < 0.05) first and increased (P < 0.05) thereafter compared with uncultured tissues. This indicated that the follicles were gradually restoring the volume ratio toward the level of the control during culture. It has also been indicated that bovine preantral follicles treated with LR3 insulinlike growth factor-I (an analog with low affinity for insulin-like growth factor binding proteins) had smaller oocyte:follicle ratios and increased oocyte degeneration compared with controls (Thomas et al., 2007); appropriate coordination of oocyte and follicle development is essential for normal oocyte growth and acquisition of developmental competence. All of these findings indicated that the development of follicles in the cultured tissues in ovo was slower when compared with the mammalian follicles in vitro, in which the number of primordial follicles was dramatically reduced with concomitant increase of developing follicles after 2 d of culture (Wandji et al., 1996; Fortune et al., 1998; Matos et al., 2007). Because the relative simpler components in vitro can easily activate follicles than in vivo, we speculated that inhibitory effects played a significant role in the maintenance of resting follicles both in ovo and in vivo; consistent with our results, ovarian grafts had slower growth than testis, thymus, liver, and spleen grafts when grafted to chicken embryo membrane (Minoura, 1921).
Figure 1. Percentage of follicles in ovarian cortical tissues of (A) 50- to 55-wk and (B) 12- to 15-wk chicken cultured in ovo for 0, 2, 4, 8, or 10 d. Bars within the same diameter lacking a common letter differ significantly (P < 0.05). Total number of follicles was, respectively, (A) 540, 520, 415, 522, and 508 and (B) 693, 468, 596, 400, and 300 on d 0, 2, 4, 8 and 10.
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Figure 2. Diameter of follicles in ovarian cortical tissues of (A) 50- to 55-wk and (B) 12- to 15-wk chicken cultured in ovo for 0, 2, 4, 8, or 10 d. Bars within the same diameter lacking a common letter differ significantly (P < 0.05). Total number of follicles was, respectively, (A) 540, 520, 415, 522, and 508 and (B) 693, 468, 596, 400, and 300 on d 0, 2, 4, 8, and 10.
Different Development of Follicles from Different-Age Chicken The volume ratio in follicles of 100 to 190 μm decreased (P < 0.05) on d 2 for both chicken ovarian pieces of 12- to 15-wk and 50- to 55-wk chicken but restored to the level of the control on, respectively, d 10 and 4. The faster restoration in 12- to 15-wk (compared with 50- to 55-wk) chicken could also be found in the follicles of other diameters (Figure 3A and B). This indicated that the follicles from chicken of 12 to 15 wk appeared more adaptive to the environment of chicken embryos, and synchronous development was achieved more quickly than those in chicken of 50 to 55 wk. We did not find similar reports stating that chicken embryos were more suitable to the survival of younger ovarian tissues. However, reports showed that in grafted bovine fetuses and ovarian cortical pieces, the primordial follicles were not activated during 10 d in ovo, and the number of healthy follicles did not decrease (Cushman et al., 2002). Because this finding seemed similar to our
results of 13- to 15-d chicken, we speculate that chicken embryos contribute more to achieving synchronous development in younger tissues. In the follicle of >200 μm, the volume ratio did not differ (P > 0.05) in the chicken of 12 to 15 wk before and after culture, but it decreased (P < 0.05) in the chicken of 50 to 55 wk on d 2 and 4 than the uncultured ones (Figure 3A and B). Meanwhile, the time to restore the volume ratios to the level of the uncultured ones shortened gradually in the follicles of <100, 100 to 190, and >200 μm in 50- to 55-wk chicken with the increase of follicular diameter (Figure 3A and B). All of these elucidated larger primary follicles survived and developed more easily than smaller ones cultured in ovo. Consistent with our results, when primary follicles of <100, 100 to 250, and >250 μm of turkey were cultured in vitro, the follicles of <250 μm could not survive more than 24 h (Bakst et al., 1998). Based on these, chicken follicles of <200 μm could survive when cultured in CAM, but difficult in vitro until now, together with maintainance of organizational integrity and normal
Figure 3. Volume ratio of oocytes and follicles in ovarian cortical tissues of (A) 50- to 55-wk and (B) 12- to 15-wk chicken cultured in ovo for 0, 2, 4, 8, or 10 d. Bars within the same diameter lacking a common letter differ significantly (P < 0.05). Total number of follicles was, respectively, (A) 400, 320, 350, 426, and 426 and (B) 540, 285, 316, 254, and 256 on d 0, 2, 4, 8, and 10.
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Table 1. Diameter of follicle or oocyte and volume ratio of oocyte and follicle in the chicken ovarian cortical pieces of 13- to 15-d chicken cultured in ovo for 0, 2, 4, or 8 d Days for culture (total number of follicles) Item
0 (174)
2 (153) a
Follicular diameter (μm) Oocyte diameter (μm) Volume ratio of oocyte and follicle
4 (144) a
45.196 ± 1.660 32.426 ± 0.863a 0.367 ± 0.012a
8 (216) a
41.140 ± 0.547 27.146 ± 1.441a 0.326 ± 0.021a
44.313 ± 1.061a 30.487 ± 1.345a 0.324 ± 0.016a
42.163 ± 1.918 27.532 ± 2.740a 0.356 ± 0.058a
a
Means within the same line with a common superscript did not differ significantly (P < 0.05).
follicular morphology in ovo, the CAM seems a preferable culture system to study early follicular development.
Development of Grafted 13- to 15-d Chicken Ovarian Cortical Tissues In Ovo The diameter of follicles, oocytes, or the volume ratios of oocyte and follicle in the ovarian cortical tissues of 13- to 15-d chicken did not differ (P > 0.05) between d 0, 2, 4, and 8 in ovo (Table 1), indicating that the CAM could not set up the growth of the oocyte or follicle. However, the follicles with incomplete or absent theca layer found in uncultured tissues were not found in the cultured tissues. All of the follicles in the cultured tissue contained theca cells (Supplemental Figure 2M to P), which indicates that culture in ovo promotes development of follicles in the ovarian pieces of 13- to 15-d chicken. However, we could not exclude whether activation of follicles could be achieved by extending culture time considering its slow courses in vivo. Most likely, an endocrine environment that contained a majority of the physiological metabolism needed for chicken ovarian development could be offered in ovo and corresponding intrinsic autocrine or paracrine could be more easily employed compared with in vitro due to the existence of blood circulation in the chicken embryo, which would make the slow development of follicles in the grafts seem more similar to those in vivo compared with in vitro.
indicated that the younger chicken ovarian pieces were more easily suitable for the environment of chicken embryo, and the differences of atretic ratios could possibly be due to differences of adaptation and reaction of grafts to the hormone and nutrient environment of embryos. Consistent with our result, Waddington et al. (1985) examined the follicular population in the ovary of birds at 46 to 49 and 70 wk of age and indicated that the number of atretic small follicles increased in old and calcium-deprived birds. The atretic ratios in grafts from 50- to 55-wk chicken increased (P < 0.05). It has also been reported that even a range of diets could affect both follicular growth and oocyte quality (Armstrong et al., 2001; Boland et al., 2001). An increasing proportion of atretic follicles was also found in cultured human ovarian tissues (Otala et al., 2002). Therefore, it seems inevitable that the grafts would be affected by the embryos. However, the atretic ratios in grafts from 12- to 15-wk chicken increased (P < 0.05) on d 2 but decreased (P < 0.05) more than 4 d compared with d 0, which is most likely due to the environmental stressors in the beginning of grafting. However, we did not find atretic follicles in the grafts of 13- to 15-d chicken. Based on these, we speculated that either the cells of follicles in older chicken ovary have become more sensitive to stimulus causing apoptosis or the young ovary developed a set of specific survival mechanisms to ensure a sufficient resting follicle supply, and the ability became gradually weakened as age increased.
Immunohistochemical Assessment of PCNA
Atretic Ratios of Follicles The ratios of atretic follicles and healthy ones in the ovarian cortical tissues were higher (P < 0.05) in the chicken of 50 to 55 wk compared with the chicken of 12 to 15 wk after being cultured in ovo (Table 2). This
Proliferating cell nuclear antigen immunoreactivity was observed in ovarian follicles at various stages of development in 12- to 15-wk, 50- to 55-wk, or 13- to 15-d chicken, and the positive expression of PCNA in the cultured tissues appeared similar to those of uncultured
Table 2. Percentage of atretic follicle in chicken ovarian cortical tissue of 50- to 55-wk and 12- to 15-wk chicken cultured in ovo for 0, 2, 4, 8, or 10 d Days for culture (d) Age of chicken (wk) 50 to 55 12 to 15 a–h
0
2 f
22.078 ± 0.376 25.926 ± 1.011e
4 c
42.735 ± 0.745 38.462 ± 1.056d
Means lacking a common superscript differ significantly (P < 0.05).
8 b
46.980 ± 0.689 18.663 ± 0.611g
10 a
56.000 ± 0.863 18.391 ± 0.717g
48.000 ± 0.685b 16.142 ± 0.630h
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Figure 4. Proliferating cell nuclear antigen immunoreactivity of healthy (A to C, E to I) or atretic follicles (D) in ovarian cortical pieces of 12- to 15-wk (A, B, D, E), 50- to 55-wk (C, F), and 13- to 15-d (G to I) chicken cultured in ovo for 0 (A, B, G), 2 (E, H), and 8 (C, D, F, I) d; bar = 100 µm. Color version available in the online PDF.
tissues, mainly in granulosa cells, oocytes, stromal cells, and some granulosa cells of various stages of follicles (Figure 4A to I), and they were positively stained (Figure 4G to I) in the theca layer of 13- to 15-d chicken ovarian tissues but rarely found in the theca layer of 12- to 15-wk and 50- to 55-wk chicken (Figure 4A to F). The PCNA is a highly conserved, auxiliary, nonhistone nuclear protein that is highly correlated with proliferation in normal cells and is usually used as a marker for proliferation (Liu et al., 1989; Hall et al., 1990; Waseem and Lane, 1990). We found that culture did not change the proliferative ability of follicular cell in ovo. A similar result was also found in cultured frozen-thawed human fetal ovarian tissue (Sadeu et al., 2006). Interestingly, most oocytes in the chicken ovary were positively stained, indicating that the oocytes were still in an active state after culture. In conclusion, the results indicated that chicken embryo could be used as the culture system for chicken ovarian cortical tissues, the follicles could survive at least 10 d and moderate healthy development was maintained, and further, the CAM system will prove useful for the study of early follicle development.
ACKNOWLEDGMENTS This investigation was financially supported by the “863” Project of China Science and Technology Ministry (2007AA100504). We thank X. Tang, L. Rei, M. Du, H. Chen, and H. Han of the Avian Reproductive Physiology and Genetic Engineering Laboratory of China Agricultural University.
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Matos, M. H. T., I. B. Lima-Verde, M. C. A. Luque, J. E. Maia Jr., J. R. V. Silva, J. J. H. Celestino, F. S. Martins, S. N. Bao, C. M. Lucci, and J. R. Figueiredo. 2007. Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro. Zygote 15:173–182. Minoura, T. 1921. A study of testis and ovary grafts on the hen’s egg and their effects on the embryo. J. Exp. Zool. 33:1–61. Nakada, K., Y. Yao, J. Mashima, M. Katoh, J. Miyazaki, and T. Hirabayashi. 1998. Skeletal muscle regeneration induced by chorioallantoic grafting. J. Muscle Res. Cell Motil. 19:169–177. Nilsson, E. E., and M. K. Skinner. 2002. Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development. Biol. Reprod. 67:1018– 1024. Otala, M., K. Erkkilä, T. Tuuri, J. Sjöberg, L. Suomalainen, V. Pentikäinen, and L. Dunkel. 2002. Cell death and its suppression in human ovarian tissue culture. Mol. Hum. Reprod. 8:228–236. Parrott, J. A., and M. K. Skinner. 1999. Kit-ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis. Endocrinology 140:4262–4271. Roig, I., R. Garcia, P. Robles, R. Cortvrindt, J. Egozcue, J. Smitz, and M. Garcia. 2006. Human fetal ovarian culture permits meiotic progression and chromosome pairing process. Hum. Reprod. 21:1359–1367. Rudnick, D. 1944. Early history and mechanics of the chick blastoderm: A review. Q. Rev. Biol. 19:187–212. Sadeu, J. C., R. Cortvrindt, R. Ron-El, E. Kasterstein, and J. Smitz. 2006. Morphological and ultrastructural evaluation of cultured frozen-thawed human fetal ovarian tissue. Fertil. Steril. 85(Suppl. 1):1130–1141. Song, Y., and F. G. Silversides. 2007a. Offspring produced from orthotopic transplantation of chicken ovaries. Poult. Sci. 86:107– 111. Song, Y., and F. G. Silversides. 2007b. Production of offspring from cryopreserved chicken testicular tissue. Poult. Sci. 86:1390– 1396. Thomas, F. H., B. K. Campbell, D. G. Armstrong, and E. E. Telfer. 2007. Effects of IGF-I bioavailability on bovine preantral follicular development in vitro. Reproduction 133:1121–1128. Waddington, D., M. M. Perry, A. B. Gilbert, and M. A. Hardie. 1985. Follicular growth and atresia in the ovaries of hens (Gallus domesticus) with diminished egg production rates. J. Reprod. Fertil. 74:399–405. Wandji, S. A., V. Srsen, A. K. Voss, J. J. Eppig, and J. E. Fortune. 1996. Initiation in vitro of growth of bovine primordial follicles. Biol. Reprod. 55:942–948. Waseem, N. H., and D. P. Lane. 1990. Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form. J. Cell Sci. 96:121–129. Webb, R., P. C. Garnsworthy, J. G. Gong, and D. G. Armstrong. 2004. Control of follicular growth: Local interactions and nutritional influences. J. Anim. Sci. 82(E. Suppl.):E63–E74. Webb, R., P. C. Garnsworthy, J. G. Gong, R. S. Robinson, and D. C. Wathes. 1999. Consequences for reproductive function of metabolic adaptation to load metabolic stress in dairy cows. Pages 99–112 in Br. Soc. Anim. Sci. Occas. Publ. No. 24. Br. Soc. Anim. Sci., Midlothian, UK. Zhan, Z., D. Ou, X. Piao, S. W. Kim, Y. Liu, and J. Wang. 2008. Dietary arginine supplementation affects microvascular development in the small intestine of early-weaned pigs. J. Nutr. 138:1304–1309.
be more easily achieved in the chicken ovarian tissues of 12- to 15-wk chicken. Neither oocyte nor granulosa cells but the thecal cells of follicles developed in grafts of 13- to 15-d chicken within 10 d of culturing in ovo, and the CAM can ease follicular atresia in chicken ovarian grafts of 12- to 15-wk chicken. The proliferative performance of follicles in the grafts was not influenced by the environment of chicken embryo. These results indicated that the chicken embryo has the ability to support slow development of primordial and primary follicles in grafted ovarian cortical tissues of chickens of different ages. The CAM system may also prove useful for the study of early follicle development with further information.
Key words: chicken, follicle development, in ovo, chicken embryo 2009 Poultry Science 88:2415–2421 doi:10.3382/ps.2008-00555
INTRODUCTION The domestic fowl provides a unique model for the study of the mechanisms involved in follicular development. The single left ovary contains follicles of various sizes and developmental stages. As in mammals, all ova within the chicken ovary are primary oocytes until shortly before ovulation (Hodges, 1974). Domestic hen ovarian follicles are organized during development, primary oocytes enclosed by the vitelline membrane become organized into a primordial follicle (up to ~80-μm diameter) after the recruitment of presumptive granulosa cells, and the perivitelline membrane is subsequently formed by granulosa cells. The initiation of primordial follicle growth to the primary follicle stage is associated with the formation of the theca layer (Johnson, 1999a,b). The constant viability and development of ovarian follicles is intricately controlled by endocrine (e.g., gonadotropins), paracrine, and autocrine factors, ©2009 Poultry Science Association Inc. Received December 20, 2008. Accepted May 16, 2009. 1 Corresponding author: [email protected]
plus neurochemical and neurohumoral factors (Johnson, 2000). Moreover, a great deal of environmental factors, such as nutrition, can influence follicular development and oocyte quality, and hence fertility (Garnsworthy and Webb, 1999; Webb et al., 1999). The activation and subsequent growth of primordial follicles still remain poorly understood. Considerable progress has been made in the culture of mammal ovarian tissue such as mouse (eppig and O’Brien, 1996; Webb et al., 2004; Gigli et al., 2005), bovine (Wandji et al., 1996; Braw-Tal and Yossefi, 1997), and human (Roig et al., 2006). In regards to reproduction of poultry, chimeras had been obtained from injection of frozen-thawed blastodermal cells (Kino et al., 1997), and offspring have been produced from orthotopic transplantation of chicken ovaries (Song and Silversides, 2007a) and cryopreserved chicken testicular tissue (Song and Silversides, 2007b). Follicles isolated from the ovary of hens have been cultured in vitro more than 5 d (Du et al., 2006). However, the manipulation was limited to the embryo and hatching chicken gonads, or the follicles were detached from the ovarian tissues. Development of a culture system for ovarian cortical tissues will contribute to our understanding for
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regulating mechanisms of follicular survival, growth, and differentiation, which are also prerequisites for the protection and use of avian reproduction resources. Grafting tissue to the chorioallantoic membrane (CAM) of chick embryos to study the development of various organs and structures is a technique that has been used by developmental biologists for decades (Rudnick, 1944; Nakada et al., 1998; Kunzi-Rapp et al., 1999; Katoh et al., 2001; Leng et al., 2004). The embryonic CAM is highly vascularized and the lack of a functional immune system before d 17 of chick embryo development prevents rejection; tissue grafted to CAM is usually rapidly vascularized. When the fetal bovine ovarian cortical pieces were placed between the CAM and the yolk sac of 6-d-old chick embryos, the primordial follicles were not activated in the grafts during 10 d in ovo; however, primordial follicles retained their ability to activate (Cushman et al., 2002). It has also been indicated that all the frozen-thawed human ovarian cortical grafts showed adhesion, and most of the follicles were healthy (Martinez-Madrid et al., 2008). However, no study has analyzed the development of follicles in the grafts in detail. Meanwhile, as the same species to the chicken embryo, no work has been done to study the supporting ability of chicken embryos to the development of chicken ovarian tissues. Therefore, ovarian cortical pieces of adult and developing chicken were grafted underneath the CAM of 6-d-old chicken embryo to examine whether the CAM model could be used as an in ovo system to culture chicken ovarian cortical tissue and to find out to what extent the grafts can be developed.
licles of around 500 μm in diameter visible to the naked eye were removed with sterile scissors. Then, the ovary was washed in warmed Dulbecco’s PBS 4 times after being in 70% alcohol for 20 s and cut into small pieces of 0.5 to 1 mm3 (Supplemental Figure 1A to C; all supplemental figures can be found online at http://ps.fass. org/content/vol88/issue11/). The cortical pieces were placed between the CAM and the yolk sac (1 piece/egg) of 6-d-old chick embryos to culture as used by Cushman et al. (2002), and the tissues were retrieved on d 0 (uncultured tissues), 2, 4, 8, and 10 (Supplemental Figure 1D to F).
Histological Analysis
Female Single Comb White Leghorn chickens of various ages were raised in floor pens. The chickens had free access to food and water. The breeder company-recommended lighting regimens were maintained throughout the experiment. Five birds of each age were slaughtered by cervical dislocation to collect ovaries on d 13 to 15, wk 12 to 15, and wk 50 to 55. Hens (in 50 to 55 wk) were kept in individual cages and oviposition time was recorded at hourly intervals in the morning and at 1700 h, and those with regular laying sequences of 6 eggs or more were selected randomly. The ovarian tissues were obtained from hens 2 h after oviposition of the first egg in the sequence. All animal experimentation was conducted in accordance with the regulations of the animal ethics committee set by China Agricultural University.
Chicken ovarian cortical pieces collected were fixed in 10% neutral buffered formalin diluted with 0.02 mol/L of PBS (8.5 mol/L of NaCl, 2.8 mol/L of Na2HPO4, and 0.4 mol/L of NaH2PO4; pH 7.2 to 7.6) for 24 h. For histological analysis, tissue samples were embedded in paraffin and sections of 5 μm were cut. Briefly, more than 8 ovarian cortical tissues from each group were processed for histological examination using the standard hematoxylin and eosin method (Supplemental Figure 1 G to I). The follicles were measured in each of 2 serial sections from the largest cross-section through the center of the ovary and averaged in each category (Parrott and Skinner, 1999; Nilsson and Skinner, 2002) and only follicles containing oocytes with a visible nucleus were measured. The sections were photographed using a Leica DFC 320 digital camera (Leica Microsystems, Wetzlar, Germany) and the diameters of follicles and oocytes were measured by Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Accordingly, 2 measurements were taken when measuring diameters and the second measurement originated at a right angle from the midpoint of the first measurement. The 2 measurements were then averaged and expressed as the diameter of the structure. The follicles in chicken ovarian pieces of 12- to 15-wk and 50- to 55-wk chicken were classified into 4 groups according to diameters: <100, 100 to 190, 200 to 290, and >300 μm. Follicles with diameters less than 100 μm were classified as primordial follicles, and more than 100 μm was considered developing follicle. The follicles with one or more of the following characters were deemed atretic follicles (such as Supplemental Figure 2I and L): a shrunken and irregularly shaped oocyte, the presence of multiple large vacuoles, the presence of blood cells or cellular debris in the ooplasm, dissociation among granulosa cells, granulosa detached from basal lamina, invasion of granulosa, loss of identity of granulosa and theca interna (Gupta et al., 1988).
Culture of Ovarian Cortical Tissues In Ovo
Immunohistochemistry
Collected ovary was transferred into sterile Dulbecco’s PBS (Sigma, St. Louis, MO); for the 12- to 15-wk and 50- to 55-wk chicken, yellow follicles and white fol-
A mouse monoclonal antibody raised against human proliferating cell nuclear antigen (PCNA; Boster, Wuhan, China) was used to detect proliferating cells
MATERIALS AND METHODS Preparation of Ovarian Cortical Pieces
CHICKEN OVARIAN TISSUE GRAFTED TO CHICKEN EMBRYO MEMBRANE
in cultured and noncultured ovarian cortical tissues. Slides were deparaffinized and rehydrated. A commercial Streptavidin-Biotin-Enzyme Complex kit (BSCXBiotech, Wuhan, China; Zhan et al., 2008) was used for immunohistochemistry. Briefly, sections were incubated in 3% H2O2 for 20 min at room temperature to block endogenous peroxidase, incubated sequentially with 5% BSA for 20 min after microwave antigen recovery, PCNA monoclonal antibody diluted 1:400 in PBS at 4°C overnight, and then corresponding biotinylated secondary antibodies against mouse and Streptavidin peroxidase. Subsequently, binding of the primary antibody was detected with diaminobenzidine. Sections were counterstained with hematoxylin. In the negative control, the primary antibodies were substituted with PBS.
Statistical Analysis Statistical analyses were performed using the GLM procedures of SAS (version 8.2, SAS Institute Inc., Cary, NC). All data are expressed as the mean ± SEM. Differences between means were determined using the Duncan’s multiple range test. A P-value <0.05 was considered statistically significant.
RESULTS AND DISCUSSION Development of Grafted 50- to 55-wk and 12- to 15-wk Chicken Ovarian Cortical Tissues In Ovo Chicken embryo maintained the healthy morphology of follicles even on d 10, and blood cells could be found as early as d 2 in ovo (Supplemental Figure 2A to P). Compared with uncultured ovarian pieces, the percentage of <100-μm follicles in tissues decreased (P < 0.05) after culture in chicken of both 50 to 55 wk (Figure 1A) and 12 to 15 wk (Figure 1B), whereas >100-μm
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follicles proved the opposite. However, smaller follicles in the ovarian cortical pieces were still more than larger follicles in the number after culture as control. The average diameter of follicles in each group did not differ nearly (P > 0.05) from each other before or after culture (Figure 2A and B) despite increasing (P < 0.05) diameter of 100- to 190-μm follicles in grafts of 12- to 15-wk chicken on d 4 than 0 (Figure 2B), which indicated that the development of follicles in the grafts was moderate. But in all the follicles in the grafts of 50to 55-wk chicken (Figure 3A), and in follicles of <300 μm in 12- to 15-wk chicken (Figure 3B), the volume ratio of oocyte and follicle decreased (P < 0.05) first and increased (P < 0.05) thereafter compared with uncultured tissues. This indicated that the follicles were gradually restoring the volume ratio toward the level of the control during culture. It has also been indicated that bovine preantral follicles treated with LR3 insulinlike growth factor-I (an analog with low affinity for insulin-like growth factor binding proteins) had smaller oocyte:follicle ratios and increased oocyte degeneration compared with controls (Thomas et al., 2007); appropriate coordination of oocyte and follicle development is essential for normal oocyte growth and acquisition of developmental competence. All of these findings indicated that the development of follicles in the cultured tissues in ovo was slower when compared with the mammalian follicles in vitro, in which the number of primordial follicles was dramatically reduced with concomitant increase of developing follicles after 2 d of culture (Wandji et al., 1996; Fortune et al., 1998; Matos et al., 2007). Because the relative simpler components in vitro can easily activate follicles than in vivo, we speculated that inhibitory effects played a significant role in the maintenance of resting follicles both in ovo and in vivo; consistent with our results, ovarian grafts had slower growth than testis, thymus, liver, and spleen grafts when grafted to chicken embryo membrane (Minoura, 1921).
Figure 1. Percentage of follicles in ovarian cortical tissues of (A) 50- to 55-wk and (B) 12- to 15-wk chicken cultured in ovo for 0, 2, 4, 8, or 10 d. Bars within the same diameter lacking a common letter differ significantly (P < 0.05). Total number of follicles was, respectively, (A) 540, 520, 415, 522, and 508 and (B) 693, 468, 596, 400, and 300 on d 0, 2, 4, 8 and 10.
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Figure 2. Diameter of follicles in ovarian cortical tissues of (A) 50- to 55-wk and (B) 12- to 15-wk chicken cultured in ovo for 0, 2, 4, 8, or 10 d. Bars within the same diameter lacking a common letter differ significantly (P < 0.05). Total number of follicles was, respectively, (A) 540, 520, 415, 522, and 508 and (B) 693, 468, 596, 400, and 300 on d 0, 2, 4, 8, and 10.
Different Development of Follicles from Different-Age Chicken The volume ratio in follicles of 100 to 190 μm decreased (P < 0.05) on d 2 for both chicken ovarian pieces of 12- to 15-wk and 50- to 55-wk chicken but restored to the level of the control on, respectively, d 10 and 4. The faster restoration in 12- to 15-wk (compared with 50- to 55-wk) chicken could also be found in the follicles of other diameters (Figure 3A and B). This indicated that the follicles from chicken of 12 to 15 wk appeared more adaptive to the environment of chicken embryos, and synchronous development was achieved more quickly than those in chicken of 50 to 55 wk. We did not find similar reports stating that chicken embryos were more suitable to the survival of younger ovarian tissues. However, reports showed that in grafted bovine fetuses and ovarian cortical pieces, the primordial follicles were not activated during 10 d in ovo, and the number of healthy follicles did not decrease (Cushman et al., 2002). Because this finding seemed similar to our
results of 13- to 15-d chicken, we speculate that chicken embryos contribute more to achieving synchronous development in younger tissues. In the follicle of >200 μm, the volume ratio did not differ (P > 0.05) in the chicken of 12 to 15 wk before and after culture, but it decreased (P < 0.05) in the chicken of 50 to 55 wk on d 2 and 4 than the uncultured ones (Figure 3A and B). Meanwhile, the time to restore the volume ratios to the level of the uncultured ones shortened gradually in the follicles of <100, 100 to 190, and >200 μm in 50- to 55-wk chicken with the increase of follicular diameter (Figure 3A and B). All of these elucidated larger primary follicles survived and developed more easily than smaller ones cultured in ovo. Consistent with our results, when primary follicles of <100, 100 to 250, and >250 μm of turkey were cultured in vitro, the follicles of <250 μm could not survive more than 24 h (Bakst et al., 1998). Based on these, chicken follicles of <200 μm could survive when cultured in CAM, but difficult in vitro until now, together with maintainance of organizational integrity and normal
Figure 3. Volume ratio of oocytes and follicles in ovarian cortical tissues of (A) 50- to 55-wk and (B) 12- to 15-wk chicken cultured in ovo for 0, 2, 4, 8, or 10 d. Bars within the same diameter lacking a common letter differ significantly (P < 0.05). Total number of follicles was, respectively, (A) 400, 320, 350, 426, and 426 and (B) 540, 285, 316, 254, and 256 on d 0, 2, 4, 8, and 10.
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CHICKEN OVARIAN TISSUE GRAFTED TO CHICKEN EMBRYO MEMBRANE
Table 1. Diameter of follicle or oocyte and volume ratio of oocyte and follicle in the chicken ovarian cortical pieces of 13- to 15-d chicken cultured in ovo for 0, 2, 4, or 8 d Days for culture (total number of follicles) Item
0 (174)
2 (153) a
Follicular diameter (μm) Oocyte diameter (μm) Volume ratio of oocyte and follicle
4 (144) a
45.196 ± 1.660 32.426 ± 0.863a 0.367 ± 0.012a
8 (216) a
41.140 ± 0.547 27.146 ± 1.441a 0.326 ± 0.021a
44.313 ± 1.061a 30.487 ± 1.345a 0.324 ± 0.016a
42.163 ± 1.918 27.532 ± 2.740a 0.356 ± 0.058a
a
Means within the same line with a common superscript did not differ significantly (P < 0.05).
follicular morphology in ovo, the CAM seems a preferable culture system to study early follicular development.
Development of Grafted 13- to 15-d Chicken Ovarian Cortical Tissues In Ovo The diameter of follicles, oocytes, or the volume ratios of oocyte and follicle in the ovarian cortical tissues of 13- to 15-d chicken did not differ (P > 0.05) between d 0, 2, 4, and 8 in ovo (Table 1), indicating that the CAM could not set up the growth of the oocyte or follicle. However, the follicles with incomplete or absent theca layer found in uncultured tissues were not found in the cultured tissues. All of the follicles in the cultured tissue contained theca cells (Supplemental Figure 2M to P), which indicates that culture in ovo promotes development of follicles in the ovarian pieces of 13- to 15-d chicken. However, we could not exclude whether activation of follicles could be achieved by extending culture time considering its slow courses in vivo. Most likely, an endocrine environment that contained a majority of the physiological metabolism needed for chicken ovarian development could be offered in ovo and corresponding intrinsic autocrine or paracrine could be more easily employed compared with in vitro due to the existence of blood circulation in the chicken embryo, which would make the slow development of follicles in the grafts seem more similar to those in vivo compared with in vitro.
indicated that the younger chicken ovarian pieces were more easily suitable for the environment of chicken embryo, and the differences of atretic ratios could possibly be due to differences of adaptation and reaction of grafts to the hormone and nutrient environment of embryos. Consistent with our result, Waddington et al. (1985) examined the follicular population in the ovary of birds at 46 to 49 and 70 wk of age and indicated that the number of atretic small follicles increased in old and calcium-deprived birds. The atretic ratios in grafts from 50- to 55-wk chicken increased (P < 0.05). It has also been reported that even a range of diets could affect both follicular growth and oocyte quality (Armstrong et al., 2001; Boland et al., 2001). An increasing proportion of atretic follicles was also found in cultured human ovarian tissues (Otala et al., 2002). Therefore, it seems inevitable that the grafts would be affected by the embryos. However, the atretic ratios in grafts from 12- to 15-wk chicken increased (P < 0.05) on d 2 but decreased (P < 0.05) more than 4 d compared with d 0, which is most likely due to the environmental stressors in the beginning of grafting. However, we did not find atretic follicles in the grafts of 13- to 15-d chicken. Based on these, we speculated that either the cells of follicles in older chicken ovary have become more sensitive to stimulus causing apoptosis or the young ovary developed a set of specific survival mechanisms to ensure a sufficient resting follicle supply, and the ability became gradually weakened as age increased.
Immunohistochemical Assessment of PCNA
Atretic Ratios of Follicles The ratios of atretic follicles and healthy ones in the ovarian cortical tissues were higher (P < 0.05) in the chicken of 50 to 55 wk compared with the chicken of 12 to 15 wk after being cultured in ovo (Table 2). This
Proliferating cell nuclear antigen immunoreactivity was observed in ovarian follicles at various stages of development in 12- to 15-wk, 50- to 55-wk, or 13- to 15-d chicken, and the positive expression of PCNA in the cultured tissues appeared similar to those of uncultured
Table 2. Percentage of atretic follicle in chicken ovarian cortical tissue of 50- to 55-wk and 12- to 15-wk chicken cultured in ovo for 0, 2, 4, 8, or 10 d Days for culture (d) Age of chicken (wk) 50 to 55 12 to 15 a–h
0
2 f
22.078 ± 0.376 25.926 ± 1.011e
4 c
42.735 ± 0.745 38.462 ± 1.056d
Means lacking a common superscript differ significantly (P < 0.05).
8 b
46.980 ± 0.689 18.663 ± 0.611g
10 a
56.000 ± 0.863 18.391 ± 0.717g
48.000 ± 0.685b 16.142 ± 0.630h
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Figure 4. Proliferating cell nuclear antigen immunoreactivity of healthy (A to C, E to I) or atretic follicles (D) in ovarian cortical pieces of 12- to 15-wk (A, B, D, E), 50- to 55-wk (C, F), and 13- to 15-d (G to I) chicken cultured in ovo for 0 (A, B, G), 2 (E, H), and 8 (C, D, F, I) d; bar = 100 µm. Color version available in the online PDF.
tissues, mainly in granulosa cells, oocytes, stromal cells, and some granulosa cells of various stages of follicles (Figure 4A to I), and they were positively stained (Figure 4G to I) in the theca layer of 13- to 15-d chicken ovarian tissues but rarely found in the theca layer of 12- to 15-wk and 50- to 55-wk chicken (Figure 4A to F). The PCNA is a highly conserved, auxiliary, nonhistone nuclear protein that is highly correlated with proliferation in normal cells and is usually used as a marker for proliferation (Liu et al., 1989; Hall et al., 1990; Waseem and Lane, 1990). We found that culture did not change the proliferative ability of follicular cell in ovo. A similar result was also found in cultured frozen-thawed human fetal ovarian tissue (Sadeu et al., 2006). Interestingly, most oocytes in the chicken ovary were positively stained, indicating that the oocytes were still in an active state after culture. In conclusion, the results indicated that chicken embryo could be used as the culture system for chicken ovarian cortical tissues, the follicles could survive at least 10 d and moderate healthy development was maintained, and further, the CAM system will prove useful for the study of early follicle development.
ACKNOWLEDGMENTS This investigation was financially supported by the “863” Project of China Science and Technology Ministry (2007AA100504). We thank X. Tang, L. Rei, M. Du, H. Chen, and H. Han of the Avian Reproductive Physiology and Genetic Engineering Laboratory of China Agricultural University.
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