In vertebrates, the reproductive system arises as bilateral anlagen, and consequently paired genital organs are commonly found in the adult. In birds, the female reproductive system is unique. Although paired anlagen appear, only the left genital primordia develop further to functional organs, except in birds of prey (Jacob and Bakst,2007).
In the chick, formation of the ovary differs between the right and left sides. In the left ovary, whose development proceeds normally, cortical cords (secondary cords) extend inward from the gonadal epithelium. From 7 days of incubation onward, the cords develop to form a cortex, whereas the medulla is composed of epithelium derived from medullary cords (primary cords) and their enlarged inner spaces (medullary lacunae). In contrast, the right ovary stops further development from 7 days onward. This is due to the absence of secondary cord formation; the right ovary has only a medulla composed of the epithelium of the medullary cord and enlarged lacunae. Eventually, the right ovary becomes smaller than the left (Romanoff,1960; Stahl and Carlon,1973; Ebensperger et al.,1988; Van Krey,1990; Ukeshima and Fujimoto,1991; Smith,2007).
Studies on bird ovaries have focused mainly on early embryological events, with emphasis on the influence of steroid hormones (Muller et al.,1979; Dorozzi et al.,1991; Elbrecht and Smith,1992) and mRNA expression profiles of genes related with sex differentiation of gonads in the chicken embryo (Yamamoto et al.,2003; Hoshino et al.,2005; Hudson et al.,2005; Ishimaru et al.,2008; Rodriguez-León et al.,2008).
Although asymmetric development of the left and right ovaries has been clearly demonstrated, few histological studies have correlated both ovaries; numerous observations have been made in the left gonad, mainly in the egg-laying hen (Johnson and Woods,2007) but not in the right one, which undergoes atrophy. Scarce information is available on the right chicken ovary, and this is mainly focused on the ultrastructure of germ (Ukeshima and Fujimoto,1991; Ukeshima,1994,1996) or epithelial cells (Avila et al.,1991) in chicken embryos and in newly hatched chickens (González-Morán,2005), but without a sequence along development. Only Brode (1928) reported gross morphological changes in the left and right ovaries from 9 days of incubation to adults in the brown Leghorn race, but there are no histomorphometric studies in both ovaries during normal development in birds.
Therefore, the aim of this study was to evaluate and correlate the histological and stereological changes in the left and right ovaries of Gallus domesticus from 8-day-old embryos to 4-week-old chickens.
MATERIALS AND METHODS
Fertile eggs from White Leghorn hens were obtained from a local supplier. Eggs were incubated at 38°C and 58.3% ± 3% relative humidity in a forced draught incubator. Some newly hatched chickens were housed in brooders at 30°C under a 14:10 light–dark cycle (lights on 06:00–20:00) with food and water ad libitum until the age of 4 weeks.
Embryos (8- and 13-day-old) and 1-day-old, 1- and 4-week-old chickens, 20 per group, were killed by decapitation, the left and right ovaries were dissected immediately and cleaned from adhesive tissue, and ovary measurements were recorded (length and width). The sex of the 8-day-old embryo to 1-day-old chickens was determined by in situ microscopic examination of the gonads.
Immediately afterward, eight left and right ovaries per group were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraplast. Cross sections (5 μm) were made and stained with hematoxylin and eosin to estimate the total volume of left and right ovaries and the total volume of cortex and medulla of the left ovary using the Cavaliere method (Howard and Reed,1998).
The other 12 left and right ovaries per group were fixed in 2.5% glutaraldehyde, washed in 0.15% phosphate buffer, pH 7.4, followed by 1% osmium tetroxide. Tissues were dehydrated in graded acetone and embedded in Epon 812. Sections (1-μm thick) were stained with toluidine blue for histological observations and stereological evaluations under light microscopy.
For the stereological study, an area of 10,000 μm2 of tissue per left and right ovaries was analyzed in all age groups. Measurements were performed in different areas, each from randomly chosen ovary sections. In each section, the total volume of all components of the ovarian medulla of left and right ovaries (blood vessels, lacunar channels, interstitial cells, and interstitium) was estimated by the volume fraction multiplied by the total volume of the medulla of left and right ovaries. Areas were determined using the Argus-20 image processor (Hammatsu, Japan).
The number of germ cells per left and right ovaries and the number of germ cells or follicles in cortex or medulla in the left ovary were obtained by multiplying the numerical density (Floderus equation) of germ cells or follicles by the total volume of medulla or cortex per ovary (Weibel and Bolender,1973).
The number of interstitial cells per left and right ovaries, volume of the individual interstitial cell, and volume of the nucleus and cytoplasm of interstitial cells were determined according to González-Morán (1997) and González-Morán and Calderon (2000).
Results were analyzed by one-way ANOVA. Differences among groups were determined by Tukey's multiple comparison test. Differences among means were considered significant at P < 0.05.
The left and right ovaries lie on the ventromedial surface of the mesonephroi, the dorsal aorta lies between them. The position of the right ovary is even more oblique than that of the left. The disparity in size between the right and left ovaries is well marked from the 8-incubation day onward, the left ovary was larger, and similar asymmetry was observed at all other ages studied (Fig. 1).
Eight-day-old chicken embryo.
At this age, the left ovary showed a cortex and medulla (Fig. 2A). The cortex consisted chiefly of a germinal epithelium constituted by several layers of somatic pyramidal cells and larger germ cells (oogonia) with clear cytoplasm. Some regions of the germinal epithelium with clusters of oogonia and some epithelial cells forming lobulations that extend from its deep layer into the medulla were observed (Fig. 2C). These correspond to the genesis of cortical cords or the cords of the second proliferation (secondary sex cords). The cortex was separated from the medulla by a thin layer of connective tissue, which forms the primary tunica albuginea.
The right ovary did not show a cortex and the germinal epithelium was flat and single layered (Fig. 3A). The ovarian medulla of the left and right ovaries was similar and presented the same elements. Observations included germ cells as solitary cells, blood vessels, lacunar channels, mesenchymal cells, and single and scarce interstitial cells with irregular surface and large irregular nuclei surrounded by scarce cytoplasm containing a few lipid droplets (Figs. 2A,E, 3A,B).
Thirteen-day-old chicken embryo.
At this age, the left ovary presented a thicker cortex than in the left ovary of 8-day-old embryos, because the germinal epithelium had several more layers of columnar cells with numerous oogonia; secondary sex cords were increased in size and number and extended into the medulla, containing more germ cells and somatic cells than in the previous age (Fig. 2B,D). The right ovary presented a flat and single-layered germinal epithelium, without a cortex (Fig. 3C) as in the previous age. The medulla of left and right ovaries presented the same components as in the previous age, but both ovaries presented a laxer tissue with more germ cells forming clusters, more developed lacunar channels, and interstitial cells arranged in small cords and some single interstitial cells (Figs. 2B,F, 3C,D). The medulla of the right ovary was much looser than that of the left ovary, the germ cell clusters were encircled by interstitial cells, and degenerating germ cells were observed within medullary lacunae (Fig. 3D).
In both ovaries, the cytoplasm of interstitial cells appeared more prominent than the nucleus, with more lipid droplets when compared with the previous age (Figs. 2F, 3D).
At this age, the left ovary showed well-differentiated regions, cortex and medulla, separated by the tunica albuginea (Fig. 3A). The secondary sex cords have separated from the germinal epithelium, forming a well-delimited superficial epithelium, reduced to a single layer of columnar cells. The cortex was thicker with large clusters of germ cells formed by both oogonia and oocytes surrounded by flattened pregranulose cells with a scanty cytoplasm (Fig. 4A,B). The right ovary had only medullary tissue with a thin germinal epithelium, formed by a single layer of flattened cells (Fig. 3E).
The left and right ovaries presented a compact medulla with moderate vasculation, and lacunar channels were enlarged. In the left ovary, the germ cells were scattered as solitary cells in the medulla, and degenerating germ cells were frequently found within the lacunar channels (Fig. 4A,C), but in the right ovary the germ cells were more abundant in the medulla than in the left ovary and formed clusters encircled by interstitial cells (Fig. 3E).
Both ovaries presented numerous interstitial cells arranged in cords of variable size and irregular shape. These cells were irregular in shape, and their cytoplasm appeared more prominent than the nucleus with abundant and large-sized lipid droplets (Figs. 3E, 4C).
In this age, the left ovary showed a thin germinal epithelium, as a single layer of cells, like in the previous age, the cortex started to interact with the subcortical medulla, the tunica albuginea, which separates the cortex and the medulla, started to regress, but the two regions can still be differentiated. The central part of the cortex presented follicles, from 34.6 to 50.0 μm in diameter, constituted by oocytes surrounded by a single layer of granulose cells to form a primordial follicle, and some interstitial cell cords and lacunar channels could be seen migrating around the primordial follicles, lacking the limit between cortex and medulla (Fig. 4D,F). However, in the extremities of the cortex, no formation of follicles was observed; only oocytes surrounded by pregranulose cells were observed (Fig. 4E). Lacunar channels were better developed in the deep medulla; among these channels, abundant cords of interstitial cells could be seen when compared with the left ovary from 1-day-old chicken (Fig. 4G). Medullary germ cells were absent; only degenerating germ cells within the medullary lacunar channels were observed (Fig. 4D).
The right ovary showed a thin germinal epithelium, as a single layer of cells, but epithelial cells were much more flattened when compared with the right ovary from 1-day-old chicken (Fig. 5A,C). The medulla was more compact with abundant interstitial cell cords; a few isolated germ cells were frequently observed within lacunar channels. A reduction of lacunar channels could be observed reducing their lumen, and the interstitial cells depicted similar characteristics to those of the medullary area of the left ovary (Fig. 5A,C).
At this age, the left ovary did not show anymore the separation of the cortex and medulla, and the ovary was well developed with very compact ovarian stromal tissue. Many primary follicles were present that remained embedded within the ovarian stromal tissue, ranging in size from 192 to 400 μm in diameter. The primary follicles stage was associated with the formation of the theca layer, which was separated from the granulose layer by the basal lamina, and remained in contact with a layer of fibroblasts. The theca was formed by lacunar channels, reducing their lumen and, in some cases, became obliterated; blood vessels and interstitial cell cords remained in the periphery of the follicle near the ovarian stroma, but the two regions of the theca were not differentiated yet (Fig. 4F). Interstitial cells were characterized by their large size and prominent cytoplasm with abundant lipid droplets when compared with the previous age (Fig. 4I).
In the right ovary, the general appearance of the germinal epithelium was similar to that of the 1-week-old chicken. The medulla was very compact, without any germ cells. Interstitial cells were very abundant, arranged in large cords, being the dominant component of the medulla. Interstitial cells presented similar characteristics to those of the previous age, showing a dramatic reduction in lacunar channels, reducing their lumen until they became obliterated (Fig. 5B,D).
Data indicate that the left ovary of chickens showed a marked increase in length, width, and total ovarian volume with age, increasing from 8-day-old chicken embryo to 4-week-old chickens, reaching the maximal increase in this last stage. Within the total ovarian volume, both the total volume of the cortex and the medulla also increased with age. In the left ovary, from 8-day-old embryo to 1-day-old chicken, the total volume occupied by the cortex was lower than that occupied by the total volume of the medulla, and in the left ovary of 1-week-old chicken, the total volume of the cortex and the medulla was similar. The right ovary of chickens showed an increase in length, width, and ovarian total volume from 8-day-old embryo to 1-day-old chicken, but diminished thereafter from 1-week-old chicken to 4-week-old chicken (Fig. 6A–C).
The left ovary of chickens showed a marked increase in the estimated total volume of all components of the ovarian medulla (blood vessels, lacunar channels, interstitial cells, and interstitium) with age. In contrast, the right ovary of chickens showed an increase only in the total volume of blood vessels, lacunar channels, and interstitium from 8-day-old chicken embryo to 1-day-old chicken, diminishing afterward until 4-week-old chickens, but the total volume of interstitial cells showed marked increases from 8-day-old embryo to 4-week-old chicken (Fig. 7A,B).
In the left and right ovaries of 8-day-old chicken embryo, the total volume occupied by the interstitial cells was lower than that occupied by the total volume of blood vessels, lacunar channels, and interstitium, but in both ovaries from 13-day-old chicken embryo to 4-week-old chicken the total volume of blood vessels was lower than that occupied by the total volume of lacunar channels, interstitial cells, and interstitium. In the left ovary from 1-day to 4-week-old chickens, the total volume of interstitial cells was higher than that occupied by blood vessels and lacunar channels, but was lower than that occupied by the total volume of the interstitium; however, in the right ovary of 1-week-old chicken, the total volume of interstitial cells was higher than that of the total volume of blood vessels and lacunar channels, and in the right ovary of 4-week-old chickens the total volume occupied by interstitial cells was higher than that of the other components of the ovarian medulla (Fig. 7A,B).
Data indicate that the total number of germ cells per left and right ovaries of chickens increased from 8-day-old chicken embryo to 1-day-old chickens, but diminished markedly in both ovaries of 1-week-old chickens; in the 4-week-old chickens, no germ cells were present (Fig. 8A). In the right ovary, all germ cells were localized in the medulla as there was no cortex, but in the left ovary, which presented cortex and medulla, the number of germ cells was larger in the medulla of the left ovary of 8-day-old chicken embryo than in the cortex. In contrast, in 13-day-old chicken embryo and 1-day-old chicken, the relationship was reversed, as there were more germ cells in the ovarian cortex than in the ovarian medulla, reaching a maximum in the ovarian cortex of 1-day-old chicken. In the ovarian medulla of 1-week-old chicken, germ cells were absent, and the ovarian cortex presented a reduction in the number of germ cells when compared with the previous age; this reduction was associated with the presence of primordial follicles in the central part of the cortex. In the left ovary of 4-week-old chicken, only primary follicles were present at this stage within the ovarian stromal tissue (Fig. 8B).
The number of interstitial cells per left ovary increased with age, but the right ovary showed an increase from 8-day-old chicken embryo to 1-day-old chicken; afterward, the number of interstitial cells decreased with age (Fig. 9A). The volume of individual interstitial cells and their cytoplasmic volume in both ovaries increased with age, reaching their maximal increase in left and right ovaries of 4-week-old chickens (Fig. 9B,C).
The present results show that the nuclear volume of the interstitial cells of both ovaries of 8-day chicken embryo was higher than the cytoplasmic volume. In contrast, from 13-day chicken embryo to 4-week-old chickens, the relationship was reversed; the cytoplasmic volume was higher than the nuclear volume (Fig. 9C).
Although asymmetric development of the left and right gonads in female chickens is clearly shown, during early development stages before sexual differentiation, chicken embryonic gonads develop symmetrically and show no obvious morphological left–right asymmetry (Smith and Sinclair,2004). Despite that the gonad is still structurally symmetric at stages 18–27 (E 3–5.5 days), left–right asymmetric gene expression occurs in the cortex in both sexes, and initial morphogenetic events occur independently from sex (Ishimaru et al.,2008).
Thereafter, the gonads display sexually dimorphic development at stages 29–30 (E 6–7 days), with bilateral testicular development in male; in females, the left cortex proliferates and develops into the ovary, whereas the right cortex disappears (Smith and Sinclair,2004). At this age, the aromatase gene is expressed only in female gonads (both left and right), but not in male gonads (Elbrecht and Smith,1992; Endo et al.,2007). The produced estrogen induces development of the ovarian cortex in the left gonad, because the estrogen receptor gene is expressed only in the epithelium of the left gonad in the female embryo (Nakabayashi et al.,1998). In the female right gonad, the cortex does not develop because of the intrinsic absence of estrogen receptor gene expression. From day 7.5 of incubation, the right ovary regresses without having receiving this hormonal stimulation, and the anatomical asymmetry becomes macroscopically evident at stage 34 (E 8 day). When the morphological asymmetry between left and right ovaries has been established already, there are few reports assessing both ovaries, most studies have focused mainly to either the left or the right ovary, during some embryonic stage or posthatching age, but without a sequence along development. In consequence, the objective of this research was to analyze and correlate the sequence of histological and stereological changes occurring in the left and right chicken ovaries (Gallus domesticus) as well as the variations in the number and size of their cells, from the start of morphological differentiation of the gonads (8-day-old chicken embryo) until the follicular development stage (4-week-old chicken).
For that reason, in this study, five specific development stages were analyzed: 8-day-old chicken embryo, when the definitive pattern of sexual differentiation and morphological asymmetry between the left and right ovaries are established (Gasc,1978); 13-day-old chicken embryo, when the hypothalamic–pituitary–gonadal axis begins to function (Woods and Weeks,1969; Jenkins and Porter,2004); 1-day-old chicken, when the left ovary shows two well-differentiated regions: cortex and medulla (González-Morán,2005); 1-week-old chicken, when the primary oocytes become organized into primordial follicles in the left ovary (González-Morán,2007); and 4-week-old chicken, when the primordial follicles grow to the primary follicles stage in the left ovary (Johnson and Woods,2007).
The results show that the disparity in size between the left and right ovaries is well marked at all examined ages; the right ovary is much smaller than the left ovary, reaching the maximal disparity in 4-week-old chickens. These results indicate a possible relation between left ovary growth and the reduction in the total volume of the right ovary. The present observations agree with Benoit (1923,1932,1950) and Domm (1927), who assumed an inhibitory influence exerted by the left ovary on the right, depressing the growth of the latter. The rudimentary gonad proliferates only in the absence of the left ovary (Kornfeld-and Nalbandov,1954; Taber and Salley,1954; Kornfeld,1958; Taber et al.,1958; Groenendijk-Huijbers,1965), but this statement deserves further research.
The histological results indicate that the right ovary lacked cortex at all examined ages; only a medulla was found. Although the left ovary showed two well-differentiated regions, cortex and medulla, the medulla region in the right ovary is very similar to that of the left ovary, having the same cellular components. These differences in size and histology between right and left ovaries confirm the asymmetric development of both gonads. These results are in agreement with previous reports indicating that, in the chicken, the formation of the ovary differs between the right and left sides (Ukeshima,1994,1996; González-Morán,2005). Some authors propose that these differences between left and right ovaries depend on the asymmetric gene expression between them, which occurred only in the cortex before sex differentiation. Asymmetric PITX2 gene expression in the left presumptive gonad leads to the asymmetric expression of the retinoic acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptor Ad4BP/SF-1, cyclin D1, and estrogen receptor α in the right presumptive gonad. As a consequence, inhibition of cell proliferation and loss of responsiveness to estrogen in the right ovarian primordium are induced (Guioli and Lovell-Badge,2007; Ishimura et al.,2008; Rodriguez-León et al.,2008). At the later stage 30, the FGF9 in the left gonad of the female is stronger than in the right gonad (Yoshioka et al.,2005). All these studies demonstrated that the differential expression of genes is involved in the process of asymmetric ovarian development in the chicken.
These evidences and the histological results of this study allow to infer that left–right asymmetric gene expression must be involved before and after gonadal sex differentiation and at all posthatching ages, and molecular mechanisms and signal transduction pathways must be specific depending on the development stage of the ovaries, to maintain the development and function of the left ovary and atrophy of the right, but more research in this sense is needed.
The histological and stereological results also demonstrate that the ovarian medulla of left and right ovaries presents the same components (blood vessels, lacunar channels, interstitial cells, and interstitium), but the proportion and organization of each component differ markedly according to whether it is the left or right ovary and the age of the animal.
It is noteworthy to mention that our observations indicate that the total volume of interstitial cells is the dominant component in the right ovary (55%) of 4-week-old chickens; cell hypertrophy is mainly responsible for the increase of total volume of interstitial cells. Previous works have detected a positive reaction for Δ5-3βHSD activity in interstitial cells in the left and right ovaries of chicken embryo and newly hatched chicken (Narbaitz and Kolodny,1964; Scheib and Haffen,1967; Samar et al.,1983; González-Morán and Calderon,2000), confirming the steroidogenic function of these cells in both ovaries. Other authors have indicated that the left and right ovaries of the chicken embryo secrete steroid hormones and respond to gonadotropins with an increase in cyclic AMP concentration and in 17β-estradiol and testosterone production (Teng and Teng,1977,1979; Teng et al.,1982); both left and right medulla express aromatase and produce estrogens (Nakabayashi et al.,1998; Endo et al.,2007), and ERα is expressed symmetrically in both sides of the medulla (Andrews et al.,1997; Smith et al.,1997), but, in a previous work, we demonstrated a cell-specific localization of ERα in the cortex and medulla of left and right ovaries (González-Morán,2005). King (1975) demonstrated that if the right ovary is removed because of disease or surgery, the left gonad hypertrophies and usually develops into a testis-like structure.
All this information indicates that the capacity of the right ovary to secrete steroid hormones is not affected by regressing, and it could be inferred that it is necessary that the right ovary must persist at all ages of development, as a medullary tissue that maintains its steroidogenic function for the normal development of the left ovary. This asymmetric development must depend on the differential expression of genes in cortex and medulla between left and right ovaries, but more experiments are needed to elicit this postulation.
In conclusion, this study suggests that there are specific age-dependent changes in left and right chicken ovaries, which could help to understand the sequence of histological and stereological events that occur in the development of the left functional ovary and the regressing of the right ovary of Gallus domesticus during normal development. There are clearly many exciting new aspects regarding molecular mechanisms and signal transduction pathways involved in the asymmetrical development of chicken ovaries to be explored.
The author thanks Biol. Sergio Rivera for his technical assistance in the experiments, and MFP. Ana Isabel Bieler Antolin and Biol. José Antonio Hernández-Gómez for their help with photographic techniques.