Journal List > J Vet Sci > v.8(3) > 1041727

Bozkurt, Aktaş, Ulkay, and Firat: Sertoli cell proliferation during the post hatching period in domestic fowl

Abstract

There has been no study aimed at directly determining of the periods of Sertoli cell proliferation in birds even domestic fowl. The aims of this study were to observe the cessation of post-hatching mitotic proliferation of Sertoli cells in domestic fowl, and to determine the volume density of Sertoli and germ cells during this period. A total of 50 Leghorn chicks were used in this study. The testes sections of the animals were immunostained with BrdU to observe the proliferation of cells from one to 10 weeks of age. The volume density of the Sertoli and germ cells were determined using the standard point counting method. The volume density of the germ cell nuclei was initially less than that of the Sertoli cells but the volume density converged by week 6, and remained relatively constant until the commencement of meiosis. Clear labeling of Sertoli and germ cells was observed from week 1 to week 7. The only those cells still labeled after 8 weeks were germ cells, indicating that Sertoli cell proliferation had ceased. Therefore, it is recommended that any research into the testes of domestic fowl should consider the cessation of Sertoli cell proliferation by approximately 8 weeks.

Introduction

Sertoli cells play an important role in determining the testis size, germ cell number and sperm production rate in adulthood [2]. The number of spermatozoa produced per day is also critically dependent on the number of Sertoli cells per testis because each Sertoli cell can support only a finite number of germ cells [13]. In all species studied so far, Sertoli cells proliferate during fetal or neonatal life as well as in the peripubertal period [16]. The period for the proliferation of Sertoli cells has been well established in certain mammals. In rats, it has been found to commence during the neonatal period and cease at 18 days of age [12]. In bulls, boars and rams, similar time spans have been observed but Sertoli cell replication has been found to last from 6 to 10 weeks [3,4,9,15]. Germ cells proliferate after a short period of quiescence and migrate from their central location towards the basal membrane, at which stage they are known as spermatogonia [10,11]. Knowledge of when Sertoli cell proliferation ceases in a species is important for planning studies and correctly interpreting the results. However, there has been no direct determination of these periods in birds, even the domestic fowl. The aims of this study were observe the cessation of post-hatching mitotic proliferation of Sertoli cells in domestic fowl, and to determine the volume density of Sertoli and germ cells during this period.

Materials and Methods

White Leghorn (Lohmann LSL) male chicks were identified by a cloacal inspection and kept under a 14Light : 10Dark lighting regime. The chicks were provided with food and water ad libitum. Five animals were selected randomly every week between one and 10 weeks of age. They were injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU, 100 mg/kg body weight), as a specific marker for cellular proliferation, and 1 h later with heparin (130 IU/kg). The chicks were then anesthetized with CO2 and perfused through the heart with a Bouin's solution using a peristaltic pump (Ismatec, Switzerland) for 10-20 min [18]. A complete color change of the liver to yellow was considered the criterion for ending perfusion. This method, cardiac perfusion with heparin, resulted in one testis not being well fixed. Other studies have used this method for rat testis perfusion with an average 99% success rate [14]. Moreover, it is easily performed and does not require experienced users.
The left testis was taken from the animals, immersed in the same fixative for 24 h and embedded in paraffin. Four-micron thick specimens were taken from the testes in a random systematic manner.
The sections were immunostained for BrdU to display the cellular proliferation. The sections were deparaffinized, rehydrated and treated with 0.3% H2O2 for 10 min in methanol. Antigen retrieval was achieved by incubating the slides with trypsin (Lab Vision, USA) for 20 min. The sections were washed with PBS (3 × 5 min) and incubated with 2 N HCl for 30 min and then with a 0.1 M borax solution for 10min. Following washing with PBS (3 × 5min), the sections were incubated for 5 min with Ultra V Block (Lab Vision, USA) to block the non-specific binding, and incubated with the anti-BrdU primary antibody (Lab Vision, USA) for 2 h at room temperature. The immunoreaction was detected using a detection kit (UltraVision; Lab Vision, USA). This involved incubating the sections with biotinylated anti-mouse immunogloblins and streptavidin peroxidase for 10 min each, and visualizing any immunoreactivity using a 3,3'-diaminobenzidine as chromagen substrate. The sections were then counterstained lightly with Mayer's hematoxylin.
In order to determine the volume density using the standard point counting method [1], testes sections from 5 animals each week were stained with Harris' hematoxylin and eosin, and observed by projecting objective images (×100) onto a monitor. Ten fields were evaluated using a systematic clock-face sampling pattern from a random starting point. This involved placing a 104-point grid printed on a transparency over each field and counting the number of grid points over the Sertoli cell nuclei, germ cell nuclei or seminiferous tubule. The total number of points over each structure, adding all 10 fields, was obtained. The total number (N) of grid points over the seminiferous tubules is expressed as the percentage of the total number of grid points (= 1040). For the Sertoli and the germ cell nuclei, the total number of grid points over each structure was divided by N and is expressed as a percentage.
An independent samples t-test was used to evaluate the differences between the mean volume density of Sertoli cell and germ cell nuclei within the same week. A one-way ANOVA test was used to examine the changes in the mean volume densities of Sertoli cells, germ cells and seminiferous tubules with time.

Results

Fig. 1 shows the clear differentiation of Sertoli cells and germ cells in a typical immature testis. The germ cells are spherical with large nuclei. The Sertoli cell nuclei are considerably smaller with an irregular shape.
BrdU labeling was strong and without a background, making it particularly suitable for studies of domestic fowl testes. Clear labelling of the Sertoli and germ cells was observed between one and seven weeks of age. However, at 8 weeks, Sertoli cell labeling was not observed in 3 fowls (Fig. 2a) but was observed in the remaining 2 fowls (Fig. 2b).
An examination of the hematoxylin stained sections at 8 weeks revealed primary spermatocytes to be present in all testes in which the Sertoli cells had not been labeled (Fig. 2d). Indeed, no primary spermatocytes were observed in any sections in which the Sertoli cells were labelled (Fig. 2c). Moreover, spermatagonia were the only cells labelled for BrdU after week 8. This suggests that the cessation of Sertoli cell replication coincides with the commencement of meiosis.
The volume density of the seminiferous tubules increased until week 8 with a minor decrease observed at weeks 2 and 5 (Fig. 3). The volume density of the germ cell nuclei was initially less than that of the Sertoli cell but these volume densities converged becoming virtually the same by week 6, and remaining relatively constant until the commencement of spermatogenesis (Fig. 4).

Discussion

Although there is no knowledge of the precise period and kinetics of Sertoli cell proliferation in domestic fowl, hemicastration studies have provided some information. Compensatory testicular hypertrophy of the testis after hemicastration has been observed to occur for up to 8 weeks of age [5,7]. Because the testis size correlates with the number of Sertoli cells number adulthood, it has simply been assumed that the Sertoli cell proliferation period occurs during the first 8 weeks post-hatching. The results from this study confirm this assumption. Therefore, it is recommended that any research involving the testes of domestic fowl should consider that Sertoli cell proliferation will have ceased by approximately 8 weeks.
When spermatocytes are observed even in single seminiferous tubules, no proliferating Sertoli cells were found anywhere else in the same testis. In rodents, Sertoli cell replication has been found to cease upon entry of the first germ cell into meiosis [17]. Sertoli cell labelling was not observed in 3 of the 5 fowls at 8 weeks. This variation between individuals suggests that the commencement of meiosis may be a more practical indicator of the cessation of Sertoli cell replication in domestic fowl.
The volume density of the nucleus is usually proportional to the number of cells. However, the volume density of the Sertoli cells was higher than that of the germ cells at week 1 and equal at week 6. This shows that germ cells proliferate faster than Sertoli cells. The commencement of meioses depends on hormonal regulation, and the number of Sertoli cells germ cells is affected and altered by steroid hormones or endocrine disruptors [6,8]. The cessation of Sertoli cell replication with the commencement of meiosis may indicate that the former may depend on a general hormonal mechanism rather than local cell signalling. However the combined effect of Sertoli and germ cells on their development should not be underestimated. These results showed that Sertoli cell proliferation ceased when the volume densities of each type of cell nucleus converged. However, more study will be needed.

Figures and Tables

Fig. 1
Section of an immature fowl testis (at age of 6 weeks) showing germ cells (arrows) and Sertoli cells (arrowhead). bar = 30 µm.
jvs-8-219-g001
Fig. 2
Testes sections of fowls at 8 weeks of age. (a) BrdU labelling only of germ cells (arrows), (b) BrdU labelling of germ cells and Sertoli cells (arrowhead), (c) Spermatocytes (stars) in the hematoxylin and eosin stained section of the animal in Fig. 2a. (d) BrdU labeling of a testis section at age 10 weeks. bar = 30 µm.
jvs-8-219-g002
Fig. 3
Changes in the volume densities of seminiferous tubule from week 1 to 8. Each point is the mean ± SE for the five birds. The differences between time groups that are identified by a different letter are significant (p < 0.001). The volume densities were estimated using standard point counting method.
jvs-8-219-g003
Fig. 4
Changes from week 1 to 8 in the volume densities of Sertoli cell and germ cell nuclei, showing the mean ± SE for the five birds. Significances of the differences in the volume densities between the Sertoli and germ cells in each week are indicated by **(p < 0.01) and ***(p < 0.001). Within the same cell groups, the differences between time groups (on the same line) that are identified by a different letter (e.g. a, b) are significant i.e. for Sertoli cells (p < 0.001) and for germ cells (p < 0.01). The volume densities were estimated using standard point counting method.
jvs-8-219-g004

Acknowledgments

The present work was supported by Research Fund of Istanbul University. Project No 264/23082004 and UDP-700/24032006.

References

1. Atanassova N, McKinnell C, Turner KJ, Walker M, Fisher JS, Morley M, Millar MR, Groome NP, Sharpe RM. Comparative effects of neonatal exposure of male rats to potent and weak (environmental) estrogen on spermatogenesis at puberty and the relationship to adult testis size and fertility: evidence for stimulatory effects of low estrogen levels. Endocrinology. 2000. 141:3898–3907.
crossref
2. Atanassova NN, Walker M, McKinnell C, Fisher JS, Sharpe RM. Evidence that androgens and oestrogens, as well as follicle-stimulating hormone, can alter Sertoli cell number in the neonatal rat. J Endocrinol. 2005. 184:107–117.
crossref
3. Berndtson WE, Igboeli G, Parker WG. The numbers of Sertoli cells in mature Holstein bulls and their relationship to quantitative aspects of spermatogenesis. Biol Reprod. 1987. 37:60–67.
crossref
4. De Reviers M, Hochereau de Reviers MT, Blanc MR, Brillard JP, Courot M, Pelletier J. Control of Sertoli and germ cell populations in the cock and sheep testis. Reprod Nutr Dev. 1980. 20:241–249.
crossref
5. De Reviers M, Williams JB. Cunningham FJ, Lake PE, Hewitt D, editors. Testis development and production of spermatozoa in the cockerel (Gallus Dometicus). Reproductive Biology of Poultry. 1984. Harlow: British Poultry Science;183–202.
6. Isobe N, Shimada M. Effects of estrone sulfate administration on reproductive function in male Japanese quail. J Poult Sci. 2003. 40:247–253.
crossref
7. Kirby D, Froman DP. Whittow GC, editor. Reproduction in Male birds. Sturkie's Avian Physiology. 2000. 5th ed. London: Academic Press;597–615.
crossref
8. Maeda T, Yoshimura Y. Effects of diethylstilbestrol administration on sperm motility and reproductive function in male Japanese quail (Coturnix japonica). J Poult Sci. 2002. 39:27–33.
crossref
9. McCoard SA, Lunstra DD, Wise TH, Ford JJ. Specific staining of Sertoli cell nuclei and evaluation of Sertoli cell number and proliferative activity in meishan and white composite boars during the neonatal period. Bio Reprod. 2001. 64:689–695.
crossref
10. McGuinness MP, Orth JM. Reinitiation of gonocyte mitosis and movement of gonocytes to the basement membrane in testes of newborn rats in vivo and in vitro. Anat Rec. 1992. 233:527–537.
crossref
11. Meehan T, Schlatt S, O'Bryan MK, de Kretser DM, Loveland KL. Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH. Dev Bio. 2000. 220:225–237.
crossref
12. Orth JM. Proliferation of Sertoli cells in fetal and postnatal rats: a quantitative autoradiographic study. Anat Rec. 1982. 203:485–492.
crossref
13. Orth JM, Gunsalus GM, Lamperti AA. Evidence from Sertoli cell-depleted rats indicates that spermatid numbers in adults depends on number of Sertoli cells produced during perinatal development. Endocrinology. 1988. 122:787–794.
crossref
14. Russell LD, Ettlin R, Hikim APS, Clegg ED, editors. Tissue preperation for evaluation of testis. Histological and Histopathological Evaluation of the Testis. 1990. Clearwater: Cache River Press;195–209.
15. Sharpe RM. Knobil E, Neill JD, editors. Regulation of spermatogenesis. Physiology of Reproduction. 1994. New York: Raven Press;1363–1434.
16. Sharpe RM, McKinnell C, Kivlin C, Fisher JS. Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. Reproduction. 2003. 125:769–784.
crossref
17. Sharpe RM, Walker M, Millar MR, Atanassova N, Morris K, McKinnell C, Saunders PTK, Fraser HM. Effect of neonatal gonadotropin-releasing hormone antagonist administration on Sertoli cell number and testicular development in the marmoset: comparison with the rat. Biol Reprod. 2000. 62:1685–1693.
crossref
18. Sprando RL, Russell LD. Spermiogenesis in the red-ear turtle (Pseudemys scripta) and the domestic fowl (Gallus domesticus): a study of cytoplasmic events including cell volume changes and cytoplasmic elimination. J Morphol. 1988. 198:95–118.
crossref
TOOLS
Similar articles