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Developmental Stages of Primary Oocytes in Turkeys
Developmental Stages of Primary Oocytes in Turkeys J. L. CARLSON,*-! M. R. BAKST,+ and M. A. OTTINGER*,2 *Department of Poultry Science, University of Maryland, College Park, Maryland 20742 and fGermplasm and Gamete Physiology Laboratory, Agricultural Research Service, USDA, Beltsville, Maryland 20705-2350 The Balbiani body was partially dispersed by Stage III (151 to 350 itm) and associated organelles appeared in clusters in the ooplasm. Golgi and SER were observed immediately subjacent to the oolemma. Stage IV oocytes (351 to 500 iim) were characterized by the absence of the Balbiani body, a more centrally located GV, and the redistribution of the mitochondria to the periphery of the oocyte. Throughout the ooplasm was vesicular SER. By Stage V (501 to 800 itm), zonation of the organelles was completed with the mitochondrial ring immediately subjacent to the oolemma and a concentric layer of lipid droplets subjacent to the mitochondrial ring. The GV was in the periphery of the oocyte. Organelle and inclusion redistribution and organelle pleomorphism were presumed to be reflective of increasing metabolic and transport requirements of the growing oocyte in the mature turkey hen.
{Key words: oocyte maturation, germinal vesicle, meiosis, oogenesis, turkey hen) 1996 Poultry Science 75:1569-1578
INTRODUCTION Marza and Marza (1935) first classified the stages of ovarian follicular maturation in the chicken as the slow growth phase, characterized by the appearance and dispersal of the Balbiani body and the initial accumulation of yolk material in the early primary follicle (0.04 to 2.0 mm in diameter), the intermediate phase, characterized by the accumulation of white yolk (2.0 to 4.0 mm), and the rapid growth phase characterized by the accumulation of yellow yolk (4.0 to 35.0 mm). Although endocrine and morphological events associated with the rapid growth phase of the hen's ovarian follicle have been documented (Marza and Marza, 1935; Perry et at., 1978a,b; Chalana and Guraya, 1979; Yoshimura et al., 1993), considerably less is known of the initial stages in the development of the early primary oocyte in the slow growth stage. Furthermore, nothing is known about the early primary oocyte in the slow growth stage of the
Received for publication February 28, 1996. Accepted for publication July 26, 1996. !Current address: University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA 19104. 2 To whom correspondence should be addressed.
turkey in egg production. Such information is necessary to better understand both the physiological basis of follicular recruitment and maturation leading to ovulation, particularly in the latter stages of egg production (Hocking et al., 1988), and the relationship between increased body weight and the subsequent decrease in egg production due to disruption to the normal follicular development (see Nestor et al., 1980; Hocking, 1992; Renema et al, 1995). Previous histological assessments of primary oocytes were limited to specimens from immature hens and were conducted with paraffin sections. Due to the thickness of the sections (6 to 8 /xm), the resolution of cells, organelles, and inclusions were limited (see reviews by Romanoff, 1960; Gilbert, 1979; Guraya, 1989). Therefore, the objective was to describe the sequential development of the turkey's primary oocyte during the early slow growth phase of follicular development in mature turkey breeders. Preliminary observations indicate that although primary oocyte development in the turkey is similar that of to other species, there are fundamental differences in the spatial distribution of organelles and inclusions and temporal events during this development that warrant a detailed separate evaluation (Carlson, 1993).
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ABSTRACT Little is known about the growth and differentiation of the primary oocyte in the sexually mature chicken or turkey hen. In this study, primary oocytes from turkey hens in egg production were examined by light and electron microscopy. Based on oocyte and germinal vesicle (GV) diameters and organelle morphology and distribution, the sequential development of the primary oocyte was divided into five stages. No Balbiani body was observed in Stage I oocytes (< 80 /xm in diameter). Pleomorphic mitochondria were localized around the GV and multivesicular bodies were scattered in the ooplasm. By Stage II (81 to 150 /xm), the Balbiani body was observed adjacent to the GV. Pleomorphic mitochondria, macrobodies, and smooth endoplasmic reticulum (SER) were associated w i t h the Balbiani b o d y . Lipid droplets w e r e predominantly localized to the periphery of the oocyte.
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MATERIALS AND METHODS
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CUE-3 Color Image Analysis system, Opelco, Sterling VA 20167.
Light Microscopy Five stages of growth and development of the primary oocyte were defined based on size and the distribution of organelles (Table 1). The Balbiani body, a dense clustering of organelles near the GV, was used as the primary marker for differentiation of the stages. The Balbiani body was absent in Stage I oocytes (Figure la). At Stage II, the Balbiani body was distinct and easily identified as a dense particulate aggregate partially surrounding the GV (Figure lb). By Stage III the Balbiani body had begun to expand and disperse (Figure lc) and by Stage IV (Figure Id) it was completely dispersed and no longer identifiable. Stage V oocytes were characterized by a distinct zoned appearance (Figure le). The percentage of cross sectional oocyte surface area occupied by the GV decreased progressively with each stage of development, with significant declines apparent between Stages II and III and Stages III and IV (Table 1). Generally, the GV was spherical and eccentrically located in the oocyte. In contrast, the profile of the GV was occasionally irregular in oocytes from the Flock B hens.
Transmission Electron Microscopy Certain ultrastructural features were selected as markers for differentiating the five stages of development. These markers included: 1) oocyte diameter; 2) changes in the ultrastructure and distribution of the organelles and inclusions; and 3) presence or absence of the Balbiani body. Mitochondria. In Stage I oocytes, mitochondria were localized around the GV and varied in appearance (Figure 2). Mitochondria were either elongated (about 1.5 to 2.0 /*m long and 0.2 fim wide) and possessed transversely and longitudinally oriented cristae or large, ovoid (about 1.3 /ma long and 0.6 ^m wide) and electron dense and randomly orientated cristae (Figure 2). [For convenience, the large, ovoid mitochondria were designated as Type 1 (Tl) and the elongated mitochondria as Type 2 (T2).] Many of the T2 mitochondria had round or angular enlarged intracristae spaces (Figure 2) and both Tl and T2 mitochondria often possessed small (0.08 /tm) dense inclusions. Stage II oocytes contained numerous Type 3 (T3) mitochondria, characterized by filament-like cristae (Figures 3, 4). At high magnification, T3 mitochondria possessed centrally located crystalloid-like cristae when viewed in cross-section (Figure 4). The ultrastructural organization of the T2 mitochondria in the Stage II oocyte varied (Figure 3) and included: defined transverse cristae; defined longitudinal cristae; no visible cristae; poorly defined random cristae; and mixtures of transverse and longitudinal cristae with some enlarged intracristae spaces. Many of the T2 mitochondria were up to 4.5 /xm in length and possessed small (0.08 /*m) dense inclusions (Figure 3).
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Ovaries from 18 Large White breeder turkeys from two commercial strains were studied. Eight hens were from the Fall 1991 flock (Flock A) and 10 hens were from the Spring 1992 flock (Flock B). All hens were 9- to 10-mo-old and in egg production for 4 to 10 wk. Hens were maintained in an environmentally controlled house on a 14:10 lightidark photoperiod and housed individually in cages. Feed and water were provided for ad libitum consumption. Hens were euthanatized by injection of pentobarbital sodium into the ulnar vein. Thin strips of ovarian cortical tissue were fixed in 0.1 M cacodylate buffered 2% paraformaldehyde (PF) plus 2.5% glutaraldehyde (GA) for a minimum of 24 h at 4 C. All specimens were then transferred to fresh fixative and cut into 1 m m 3 pieces. Fixed samples were rinsed in 0.1 M cacodylate buffer or Millonig's phosphate buffer plus 5% sucrose for 30 min, followed by two 10-min rinses in buffer, then stored in buffer overnight at 4 C. All tissue was then postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 30 min followed by two 10-min washings in 0.1 M cacodylate buffer or Millonig's phosphate buffer. After the last rinse they were stored in buffer overnight at 4 C and then dehydrated in ethanol (5 min each in 50%, 70%, 85%, 95% and then three 30-s changes in 100% ethanol). Dehydrated samples were placed in 1:1 mixture of a low viscosity embedding medium and ethanol for 10 min followed by 2:1 mixture of embedding medium and ethanol for 15 min and then 100% embedding medium for 15 min. Embedding was performed under low vacuum at room temperature for 4 h. The resin was then cured at 68 C for 48 h. One-micrometer-thick serial sections were cut on an ultramicrotome with glass knives. Sections were mounted on slides and stained with toluidine blue for examination by light microscopy (LM). Each oocyte was sectioned until the central region of the germinal vesicle (GV) was reached. The tissue blocks were then trimmed and 90-nm sections were cut with a diamond knife on an ultramicrotome. These sections were collected on copper grids and stained for 30 min in ethanolic uranyl acetate and 10 min in lead citrate. The sections were examined with a transmission electron microscope (TEM) operated at 75 kV. Oocyte diameter and GV/oocyte area measurements were made on the 1 jum, toluidine-stained sections using an image analysis system. 3 Data for each stage are presented as the mean ± standard error of the mean. Significant differences between stages was determined by approximate t test for means of samples of unequal sample size and unequal variance at P < 0.05 (Sokal and Rohlf, 1981).
RESULTS
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Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on March 15, 2015 FIGURE 1. a) A light micrograph of a Stage 1 oocyte characterized by a prominent germinal vesicle. Organelles are sparse in the ooplasm. The granulosa cell layer and its pronounced basement membrane are also evident. (Bar = 30 pm) b) A light micrograph of a Stage II oocyte characterized by a prominent germinal vesicle surrounded by organelles forming the Balbiani body. Lipid is observed in the periphery of the oocyte. (Bar = 17 ^m) c) A light micrograph of a Stage III oocyte characterized an increase in oocyte diameter and dispersal of the Balbiani body. The germinal vesicle is in a more eccentric position. (Bar = 60 /mi).
Stage II oocytes were characterized by the initial dispersal of the Balbiani body and the subsequent distribution of lipid throughout the ooplasm (Figure 5a). Type 2 and T3 mitochondria were located in clusters of
organelles throughout the ooplasm in the Stage III oocyte (Figure 5b). Type 2 mitochondria had transversely or randomly oriented cristae, often with enlarged intracristae spaces. By Stage III, the ultrastructure of T2 mitochon-
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dria was quite variable and included elongated rodshaped, branched, dumbbell, and donut configurations (Figure 6). Small, round inclusions were found in all types of mitochondria but were most prevalent in the T3 mitochondria. The two characteristics that differentiated Stage IV and V oocytes from Stage II and III oocytes were the absence of T3 mitochondria and the peripheral redistribution of the mitochondria in the Stage IV and V oocytes (Figures 7, 8). The latter thus formed a region referred to as the mitochondrial ring. The pleomorphic ultrastructural appearance of T2 mitochondria remained unchanged (Figures 7, 8, 10). Smooth Endoplasmic Reticulum. In Stage II oocytes, vesicular and more elongated cisterna of SER were primarily associated with the mitochondria in the Balbiani body (Figure 3). In Stage III oocytes, elongated SER was found aligned along the oolemma and nuclear envelop (Figures 5a,b). Also prevalent were vesicular SER, which were associated with mitochondria throughout the oocyte (Figure 6). In Stage IV and V oocytes, the vesicular SER was dispersed throughout the ooplasm (Figures 8, 10). Additional Ooplasmic Components. The ooplasm of Stage I oocytes contained multivesicular bodies (Figure 2) and macrobodies. Golgi were observed in the Balbiani body and in the cortical region in Stage II oocytes (Figure 9) and in the ooplasm of Stage III, IV, and V oocytes (Figures 5a, 7). Golgi subjacent to the oolemma were oriented with the maturing (concave) face in the direction of the granulosa cell layer. Macrobodies (Figure 3), which were scattered throughout the ooplasm, and microbodies were common in the
Stage II oocytes. Lipid droplets and a few yolk granules appeared to be more concentrated in the cortical region of the oocyte (Figure 9). Lipid droplets, macrobodies, and multivesicular bodies were observed throughout the Stage III oocytes (Figures 5a, b). Golgi were located within the clusters of mitochondria near the GV or in the cortical region with their maturing face oriented towards the center of the oocyte. In Stage IV oocytes, macrobodies had increased in number and multivesicular bodies appeared to decrease in number when compared to previous stages. The maturing face of the Golgi adjacent to the oolemma appeared to be randomly oriented. Vesicular SER was located adjacent to the nuclear envelop (Figure 8). In Stage V oocytes, yolk granules and macrobodies were scattered throughout the ooplasm (Figure 10). Many of the macrobodies possessed transosomes and had the appearance of yolk granules. Lipid droplets were concentrated in a concentric layer subjacent to the mitochondrial ring. At no stage in the development of the turkey primary oocyte was a yolk nucleus observed.
DISCUSSION This study defined the sequence of morphological and ultrastructural changes of primary oocytes in the slow growth phase of follicular development in mature turkey breeder hens. This description has not been reported previously and is important given the continued interest in elucidating both the physiological basis of follicular recruitment, particularly as the turkey ages (Hocking et al., 1988), and the relationship between
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FIGURE 1 (continued), d) A light micrograph of a Stage IV oocyte with lipid distributed in its cortical region. The Balbiani body is fully dispersed. (Bar = 67 pm). e) A light micrograph of a Stage V oocyte illustrating the zone-like distribution of organelles within the oocyte. Note the Stage I oocytes. (Bar = 108 /art).
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Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on March 15, 2015 FIGURE 2. A dense cluster of mitochondria (arrowheads and arrows) is an indication of the future Balbiani body in the Stage I oocyte. Large, ovoid, Type 1 mitochondria (arrows) and elongated Type 2 mitochondria (arrowheads) are apparent. Triangular-shaped intracristae spaces are also observed in some Type 2 mitochondria (arrow #2). Also observed are several multivesicular bodies (MVB) adjacent to the germinal vesicle (GV). (Bar = 1.4 ^m). FIGURE 3. In Stage II oocytes, mitochondria typically had a pleomorphic appearance. Type 2 mitochondria remained elongated but the orientation of their cristae was quite variable (arrowheads). Type 3 mitochondria, which were characterized by their filament-like cristae, were observed in Stage II oocytes (arrows; T3). Also note that the arrowheads are directed toward small dense inclusions in the mitochondrial matrix. A microbody (MB) and vesicular smooth endoplasmic reticulum (SER; thick arrow) and more elongated SER cisterna (arrow outline) are also observed. (Bar = 1.0 /mi). FIGURE 4. A cross section of a Type 3 mitochondrion (arrow) revealed the detailed configuration and filamentous appearance of its cristae. Type 2 mitochondria are also observed. (Bar = 2.4 /*m).
increased body weight and the subsequent decrease in egg production due to disruption to the normal follicular development (see Nestor et ah, 1980; Hocking, 1992; Renema et al., 1995). Whether this staging procedure is applicable to the growth and development of primary oocytes in immature turkey hens is not yet known.
In mature turkey hens, development of primary oocytes between 50 and 800 jiim in diameter can be divided into five stages based on its diameter, and the morphology, ultrastructure, and distribution of organelles and inclusions. Previously, staging of the avian primary oocyte from the pre-Balbiani stage to the stage characterized by zonation of the organelles and inclu-
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FIGURE 5. a) In this Stage III oocyte, smooth endoplasmic reticulum (SER) (arrow) and a Type 3 (T3) mitochondrion (arrow 3) are adjacent to the nuclear envelop (arrowheads). Immediately above this Type 3 mitochondrion is a dumbbell shaped Type 2 mitochondrion. A Golgi body (G) and several lipid droplets (L) are also observed. (Bar = 2.4 ^m). b) A complex array of SER (arrow) is adjacent to the oolemma (arrowhead), which separates the oocyte from the granulosa cell layer (portions of granulosa cells are observed). Several elongated Type 2 (arrow 2) and Type 3 (arrow 3) mitochondria are also present in this cluster of organelles. (Bar = 2.4 fim).
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sions was based on a composite of several different species often of undocumented reproductive status. These species included the chicken (Gallus gallus), sparrow (Passer domesticus), pigeon (Columba livid), crow (Corvus splendens), myna (Acridotheres tristis), and the swallow (Hirundo rustica) or other feral species (see Romanoff, 1960; Guraya, 1989). Although fundamentally similar to other avian species, significant differences in the characteristics of the turkey primary oocytes compared to that of the primary oocytes of other species, particularly the chicken, were noted. Unlike in the chicken oocyte, lipid droplets did not aggregate around the GV opposite the Balbiani body in turkey oocytes. Furthermore, whereas lipid droplets were redistributed to the periphery in 170 to 260 /xm diameter oocytes in the chicken, a similar redistribution of lipid droplets in the turkey oocyte was not observed until Stage IV (351 to 500 [im in diameter). Golgi, which were a prominent feature in the early chicken oocyte (reviewed by Romanoff, 1960), were not a significant feature in the early primary oocyte of the turkey. Finally, a yolk-nucleus, which Guraya (1989) defined as
TURKEY PRIMARY OOCYTES
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FIGURE 6. Organelles in the dispersing Balbiani body of a Stage III oocyte include branching and dumbbell-shaped mitochondria (arrows). Macrobodies are evident (arrowheads) as is vesicular smooth endoplasmic reticulum (SER) (thick arrow) in the vicinity of the nuclear envelop (curved arrow). Variable amounts of vesicular SER were observed throughout the ooplasm in Stage II through Stage V oocytes. (Bar = 2.0 (im). FIGURE 7. Pleomorphic Type 2 mitochondria are in a more cortical position in the Stage V oocyte and form the mitochondrial ring. Granulosa cell extensions project into the ooplasm (arrowheads) and some appear to be enveloped by dilated vesicular smooth endoplasmic reticulum (black arrows). Golgi (white arrows) are also observed. (Bar = 2.5 /mi).
a component of the Balbiani body in the chicken oocyte involved in the multiplication of mitochondria, Golgi, and lipid droplets, was not observed in the turkey primary oocyte. It is suggested that the Stage I primary oocyte in the turkey was quiescent and that formation of the Balbiani body, which characterized the Stage II primary oocyte, was a manifestation of "follicular recruitment". This possibility is supported by Guraya's (1989) suggestion that the Balbiani body was the site of high metabolic activity, a prerequisite for further growth and development. The movement of organelles and inclusions during Balbiani body dispersal (Stage III) away from the more centrally located GV correlated with an increase in the growth of the Stage III oocyte relative to the GV. Such
observations suggest that the redistribution of organelles to the periphery of the primary oocyte has a functional role with regard to the transport of yolk precursor materials into the oocyte and possibly yolk granule formation. Likewise, the abundance of the pleomorphic T2 mitochondria, particularly the dumbbell configuration, is indicative of mitochondrial replication, a necessary prerequisite for oocyte growth and development. Although intramitochondrial yolk-crystals have been described in frog oocytes (Massover, 1971), the filamentous component of T3 mitochondria has not previously been described in either avian or amphibian oocytes (Schjeide et al., 1963a,b, 1966; Greenfield, 1966; Bellairs, 1967; Thiaw and Mattei, 1992; Kovacs et al, 1992). However, a filamentous component in mitochondria has been described in guinea pig oocytes (Anderson, 1974)
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Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on March 15, 2015 FIGURE 8. In the vicinity of the germinal vesicle in this Stage IV oocyte are elongated Type 2 mitochondria (arrows), macrobodies (arrowheads), and an abundance of vesicular smooth endoplasmic reticulum (thick arrows). The nuclear envelop (curved arrow) is observed. (Bar = 4.0 p.m). FIGURE 9. In this Stage II oocyte, the Golgi (white arrows), which are adjacent to the oolemma, are associated with vesicular smooth endoplasmic reticulum (thick arrow). Lipid droplets (arrow outlines) and yolk granules (double headed arrow) are also observed. Note the granulosa cells. (Bar = 2.2 iaa). FIGURE 10. Macrobodies (arrowheads) and yolk granules (YG) are observed in a Stage V oocyte. Some macrobodies have formed a dark central area (arrow) rendering them similar in appearance to the yolk granules. The vesicular smooth endoplasmic reticulum is dispersed throughout the ooplasm as are lipid droplets (arrow outline). (Bar = 2.8 jitm).
and neoplastic oncocytes (Tandler and Hoppel, 1972). In a review of oocyte ultrastructure, Anderson (1974) noted the paucity of SER in vertebrate oocytes. With its abundant vesicular SER, the turkey primary oocyte appears to be an exception. Likewise, rough endoplasmic reticulum, which was commonly observed in oocytes from other species (Anderson, 1974), was notably absent from the turkey's primary oocytes.
The close proximity of SER with the oolemma and Golgi in Stages II through Stage V suggests that the SER is involved in plasma membrane synthesis. It has been shown that SER is capable of synthesizing ceramides, which are transported to the Golgi to serve are a precursor for the synthesis of glycosphingolipids and sphingomyelins, both of which are components of the plasma membrane (Alberts et ah, 1989). Furthermore, in
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An understanding of the normal growth and development of the primary oocyte will contribute to our fundamental understanding of the oocyte maturation and the possible influence the primary oocyte plays in follicular recruitment and aberrant multiple hierarchy formation. In addition, the staging procedure described in this study will provide objective criteria defining the magnitude of maturation of isolated, in vitro matured turkey oocytes.
ACKNOWLEDGMENTS This paper is Scientific Article Number A7738; Contribution Number 9058 of the Maryland Agriculture Experiment Station (Department of Poultry Science). Research conducted by J. Carlson was in partial fulfillment of requirements for the Master's Degree.
REFERENCES Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts, and J. Watson, 1989. Molecular Biology of The Cell. Garland Publishing, Inc., New York, NY. Anderson, E., 1974. Comparative aspects of the ultrastructure of the female gamete. Pages 1-70 in: International Review of Cytology. G. H. Bourne and J. F. Danielli, ed. Academic Press, New York, NY. Bellairs, R., 1967. Aspects of the development of yolk spheres in the hen's oocyte, studied by electron microscopy. J. Embryol. Exp. Morphol. 17:267-281. Bubel, A., 1989. Microstructure and Function of Cells. Electron Micrographs of Cell Ultrastructure. Halsted Press, New York, NY. Carlson, J. L., 1993. Characteristics and morphology of developing yolkless oocytes in turkeys. Master's thesis. University of Maryland, College Park, MD. Chalana. R. K., and S. S. Guraya, 1979. Morphological and histochemical observations on the primordial and early growing oocytes of crow (Corvus splendens) and myna (Acridotheres tristis). Poultry Sci. 58:225-231. Gilbert, A. B., 1979. Female genital organs. Pages 237-360 in: Form and Function in Birds. A. S. King and J. McLelland, ed. Academic Press, London, UK. Greenfield, M. L., 1966. The oocytes of the domestic chicken shortly after hatching, studied by electron microscopy. J. Embryol. Exp. Morphol. 15:293-316. Guraya, S. S., 1989. Ovarian Follicles in Reptiles and Birds. Springer-Verlag, New York, NY. Hocking, P. M., 1992. Genetic and environmental control of ovarian function in turkeys at sexual maturity. Br. Poult. Sci. 33:437-448. Hocking, P. M., A. B. Gilbert, C. C. Whitehead, and M. A. Walker, 1988. Effects of age and early lighting on ovarian function in breeding turkeys. Br. Poult. Sci. 29:639-647. Kovacs, J., V. Forgo, and P. Peczely, 1992. The fine structure of the follicular cells in growing and atretic ovarian follicles of the domestic goose. Cell Tiss. Res. 267:561-569. Marza, V. D., and E. V. Marza, 1935. The formation of the hen's egg. Parts I-IV. Q. J. Microsc. Sci. 78:183-249. Massover, W. H., 1971. Intramitochondrial yolk crystals of frog oocytes II. Expulsion of intramitochondrial yolk crystals to form single membrane bound hexagonal crystalloids. J. Ultrastruct. Res. 36:603-620.
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pancreatic exocrine cells, the transfer of membranebound vesicles from the Golgi to the plasma membrane is thought to be responsible for the addition of surface plasma membrane during growth (Bubel, 1989), a mechanism that should be functioning in the growing oocyte. In Stage I oocytes, the Golgi had the maturing (concave) face oriented toward the GV. One of the functions of Golgi is synthesis of peptide signaling molecules (Alberts et al, 1989). Possibly in the Stage I oocytes, the Golgi may have been involved in synthesis of a signaling molecule that signaled the initiation of growth. In Stage II oocytes, larger, more well developed Golgi were found primarily in the cortical region of the oocyte and oriented with their maturing faces toward the granulosa cell layer. This change in orientation may reflect a plasma membrane synthesizing function in the immediate vicinity of the oolemma; however, in Stage IV and V oocytes, the orientation of the Golgi was more random. As noted above, the oolemma was becoming increasingly convoluted and the surface area is increasing, both of which not only necessitate the de novo synthesis of plasma membrane but an increase in the lipid and protein transport capacity across the oolemma. It may also reflect some activity of yolk granule formation. Macrobodies [membrane-bound clusters of transosomes surrounded by a common membrane (Greenfield, 1966)] and multivesicular bodies [membrane-bound vesicles that contain small vesicles, as defined by Bubel (1989)] play an important role in the initial development of certain types of yolk spheres (Greenfield, 1966; Bellairs, 1967; Paulson and Rosenberg, 1972; Schjeide et al, 1974). Although Paulson and Rosenberg (1972) indicated that yolk sphere formation began when the chicken primary oocyte was 2.5 to 3.0 mm in diameter, a few yolk granules were observed at all five stages of primary oocyte development in the turkey. An unexpected finding during LM examination of the primary oocytes was the difference in the number of primary oocytes having irregular GV between the Flocks A and B. All hens used for this study were Large White turkeys obtained from the same commercial source. However, the Flock A was a female line (selected for egg production) and Flock B was a male line (selected for high body weight) and consequently produce fewer eggs, possibly as a result of disruption in the development of the normal follicular hierarchy (see Nestor et al, 1980; Hocking, 1992; Renema et al, 1995). The male-line hens, which are selected for increased body weight and consequently suffer from reduced egg production, had a higher incidence of irregular GV than the oocytes of hens selected for egg production. If GV irregularity presages follicular atresia, it can be speculated that decreased egg production by Large White male-line hens may be due to a greater percentage of the primary oocytes undergoing follicular atresia than with hens genetically selected for egg production.
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CARLSON ET Ah. Origins and roles of mitochondria-like organelles. Growth 27:111-124. Schjeide, O. A., R. G. McCandless, and R. J. Munn, 1963b. Possible participation of RNA in formation of mitochondria-like organelles. Growth 27:125-128. Schjeide, O. A., R. J. Munn, R. G. McCandless, and R. Edwards, 1966. Unique organelles of avian oocytes. Growth 30: 471^89. Schjeide, O. A., L. Hanzely, S. J. Holshouser, and W. E. Briles, 1974. Production and fates of unique organelles (transosomes) in ovarian follicles of Gallus domesticus under various conditions. Cell Tiss. Res. 156:47-59. Sokal, R. R., and F. J. Rohlf, 1981. Biometry. W. H. Freeman and Co., New York, NY. Tandler, B., and C. L. Hoppel, 1972. Mitochondria. Academic Press, New York, NY. Thiaw, O. T., and X. Mattei, 1992. Natural degenerating mitochondria in ovarian follicles of a cyprinodontidae fish, Epiplatys spilargyreus (Teleost). Mol. Reprod. Dev. 32: 67-72. Yoshimura, Y., T. Okamoto, and T. Tamura, 1993. Ultrastructural changes of oocyte and follicular wall during oocyte maturation in the Japanese quail (Coturnix coturnix japonica). J. Reprod. Fertil. 97:189-196.
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Nestor, K. E., W. C. Bacon, and P. A. Renner, 1980. The influence of genetic changes in total egg production, clutch length, broodiness, and body weight on ovarian follicular development in turkeys. Poultry Sci. 59:1694-1699. Paulson, J. L., and M. D. Rosenberg, 1972. The function and transposition of lining bodies in developing avian oocytes. J. Ultrastructure Res. 40:25-43. Perry, M. M., A. B. Gilbert, and A. J. Evans, 1978a. The structure of the germinal disc region of the hen's ovarian follicle during the rapid growth phase. J. Anat. 127: 379-392. Perry, M. M., A. B. Gilbert, and A. J. Evans, 1978b. Electron microscope observations on the ovarian follicle of the domestic fowl during the rapid growth phase. J. Anat. 125: 481-497. Renema, R. A., F. E. Robinson, V. L. Melnychuk, R. T. Hardin, L. G. Bagley, D. A. Emmerson, and J. R. Blackman, 1995. The use of feed restriction for improving reproductive traits in male-line large white turkey hens. 2. Ovary morphology and laying traits. Poultry Sci. 74:102-120. Romanoff, A. L., 1960. The Avian Embryo. Macmillan, New York, NY. Schjeide, O. A., R. G. McCandless, and R. J. Munn, 1963a. Further observations on the developing avian oocyte.
{Key words: oocyte maturation, germinal vesicle, meiosis, oogenesis, turkey hen) 1996 Poultry Science 75:1569-1578
INTRODUCTION Marza and Marza (1935) first classified the stages of ovarian follicular maturation in the chicken as the slow growth phase, characterized by the appearance and dispersal of the Balbiani body and the initial accumulation of yolk material in the early primary follicle (0.04 to 2.0 mm in diameter), the intermediate phase, characterized by the accumulation of white yolk (2.0 to 4.0 mm), and the rapid growth phase characterized by the accumulation of yellow yolk (4.0 to 35.0 mm). Although endocrine and morphological events associated with the rapid growth phase of the hen's ovarian follicle have been documented (Marza and Marza, 1935; Perry et at., 1978a,b; Chalana and Guraya, 1979; Yoshimura et al., 1993), considerably less is known of the initial stages in the development of the early primary oocyte in the slow growth stage. Furthermore, nothing is known about the early primary oocyte in the slow growth stage of the
Received for publication February 28, 1996. Accepted for publication July 26, 1996. !Current address: University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA 19104. 2 To whom correspondence should be addressed.
turkey in egg production. Such information is necessary to better understand both the physiological basis of follicular recruitment and maturation leading to ovulation, particularly in the latter stages of egg production (Hocking et al., 1988), and the relationship between increased body weight and the subsequent decrease in egg production due to disruption to the normal follicular development (see Nestor et al., 1980; Hocking, 1992; Renema et al, 1995). Previous histological assessments of primary oocytes were limited to specimens from immature hens and were conducted with paraffin sections. Due to the thickness of the sections (6 to 8 /xm), the resolution of cells, organelles, and inclusions were limited (see reviews by Romanoff, 1960; Gilbert, 1979; Guraya, 1989). Therefore, the objective was to describe the sequential development of the turkey's primary oocyte during the early slow growth phase of follicular development in mature turkey breeders. Preliminary observations indicate that although primary oocyte development in the turkey is similar that of to other species, there are fundamental differences in the spatial distribution of organelles and inclusions and temporal events during this development that warrant a detailed separate evaluation (Carlson, 1993).
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ABSTRACT Little is known about the growth and differentiation of the primary oocyte in the sexually mature chicken or turkey hen. In this study, primary oocytes from turkey hens in egg production were examined by light and electron microscopy. Based on oocyte and germinal vesicle (GV) diameters and organelle morphology and distribution, the sequential development of the primary oocyte was divided into five stages. No Balbiani body was observed in Stage I oocytes (< 80 /xm in diameter). Pleomorphic mitochondria were localized around the GV and multivesicular bodies were scattered in the ooplasm. By Stage II (81 to 150 /xm), the Balbiani body was observed adjacent to the GV. Pleomorphic mitochondria, macrobodies, and smooth endoplasmic reticulum (SER) were associated w i t h the Balbiani b o d y . Lipid droplets w e r e predominantly localized to the periphery of the oocyte.
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MATERIALS AND METHODS
3
CUE-3 Color Image Analysis system, Opelco, Sterling VA 20167.
Light Microscopy Five stages of growth and development of the primary oocyte were defined based on size and the distribution of organelles (Table 1). The Balbiani body, a dense clustering of organelles near the GV, was used as the primary marker for differentiation of the stages. The Balbiani body was absent in Stage I oocytes (Figure la). At Stage II, the Balbiani body was distinct and easily identified as a dense particulate aggregate partially surrounding the GV (Figure lb). By Stage III the Balbiani body had begun to expand and disperse (Figure lc) and by Stage IV (Figure Id) it was completely dispersed and no longer identifiable. Stage V oocytes were characterized by a distinct zoned appearance (Figure le). The percentage of cross sectional oocyte surface area occupied by the GV decreased progressively with each stage of development, with significant declines apparent between Stages II and III and Stages III and IV (Table 1). Generally, the GV was spherical and eccentrically located in the oocyte. In contrast, the profile of the GV was occasionally irregular in oocytes from the Flock B hens.
Transmission Electron Microscopy Certain ultrastructural features were selected as markers for differentiating the five stages of development. These markers included: 1) oocyte diameter; 2) changes in the ultrastructure and distribution of the organelles and inclusions; and 3) presence or absence of the Balbiani body. Mitochondria. In Stage I oocytes, mitochondria were localized around the GV and varied in appearance (Figure 2). Mitochondria were either elongated (about 1.5 to 2.0 /*m long and 0.2 fim wide) and possessed transversely and longitudinally oriented cristae or large, ovoid (about 1.3 /ma long and 0.6 ^m wide) and electron dense and randomly orientated cristae (Figure 2). [For convenience, the large, ovoid mitochondria were designated as Type 1 (Tl) and the elongated mitochondria as Type 2 (T2).] Many of the T2 mitochondria had round or angular enlarged intracristae spaces (Figure 2) and both Tl and T2 mitochondria often possessed small (0.08 /tm) dense inclusions. Stage II oocytes contained numerous Type 3 (T3) mitochondria, characterized by filament-like cristae (Figures 3, 4). At high magnification, T3 mitochondria possessed centrally located crystalloid-like cristae when viewed in cross-section (Figure 4). The ultrastructural organization of the T2 mitochondria in the Stage II oocyte varied (Figure 3) and included: defined transverse cristae; defined longitudinal cristae; no visible cristae; poorly defined random cristae; and mixtures of transverse and longitudinal cristae with some enlarged intracristae spaces. Many of the T2 mitochondria were up to 4.5 /xm in length and possessed small (0.08 /*m) dense inclusions (Figure 3).
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Ovaries from 18 Large White breeder turkeys from two commercial strains were studied. Eight hens were from the Fall 1991 flock (Flock A) and 10 hens were from the Spring 1992 flock (Flock B). All hens were 9- to 10-mo-old and in egg production for 4 to 10 wk. Hens were maintained in an environmentally controlled house on a 14:10 lightidark photoperiod and housed individually in cages. Feed and water were provided for ad libitum consumption. Hens were euthanatized by injection of pentobarbital sodium into the ulnar vein. Thin strips of ovarian cortical tissue were fixed in 0.1 M cacodylate buffered 2% paraformaldehyde (PF) plus 2.5% glutaraldehyde (GA) for a minimum of 24 h at 4 C. All specimens were then transferred to fresh fixative and cut into 1 m m 3 pieces. Fixed samples were rinsed in 0.1 M cacodylate buffer or Millonig's phosphate buffer plus 5% sucrose for 30 min, followed by two 10-min rinses in buffer, then stored in buffer overnight at 4 C. All tissue was then postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 30 min followed by two 10-min washings in 0.1 M cacodylate buffer or Millonig's phosphate buffer. After the last rinse they were stored in buffer overnight at 4 C and then dehydrated in ethanol (5 min each in 50%, 70%, 85%, 95% and then three 30-s changes in 100% ethanol). Dehydrated samples were placed in 1:1 mixture of a low viscosity embedding medium and ethanol for 10 min followed by 2:1 mixture of embedding medium and ethanol for 15 min and then 100% embedding medium for 15 min. Embedding was performed under low vacuum at room temperature for 4 h. The resin was then cured at 68 C for 48 h. One-micrometer-thick serial sections were cut on an ultramicrotome with glass knives. Sections were mounted on slides and stained with toluidine blue for examination by light microscopy (LM). Each oocyte was sectioned until the central region of the germinal vesicle (GV) was reached. The tissue blocks were then trimmed and 90-nm sections were cut with a diamond knife on an ultramicrotome. These sections were collected on copper grids and stained for 30 min in ethanolic uranyl acetate and 10 min in lead citrate. The sections were examined with a transmission electron microscope (TEM) operated at 75 kV. Oocyte diameter and GV/oocyte area measurements were made on the 1 jum, toluidine-stained sections using an image analysis system. 3 Data for each stage are presented as the mean ± standard error of the mean. Significant differences between stages was determined by approximate t test for means of samples of unequal sample size and unequal variance at P < 0.05 (Sokal and Rohlf, 1981).
RESULTS
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Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on March 15, 2015 FIGURE 1. a) A light micrograph of a Stage 1 oocyte characterized by a prominent germinal vesicle. Organelles are sparse in the ooplasm. The granulosa cell layer and its pronounced basement membrane are also evident. (Bar = 30 pm) b) A light micrograph of a Stage II oocyte characterized by a prominent germinal vesicle surrounded by organelles forming the Balbiani body. Lipid is observed in the periphery of the oocyte. (Bar = 17 ^m) c) A light micrograph of a Stage III oocyte characterized an increase in oocyte diameter and dispersal of the Balbiani body. The germinal vesicle is in a more eccentric position. (Bar = 60 /mi).
Stage II oocytes were characterized by the initial dispersal of the Balbiani body and the subsequent distribution of lipid throughout the ooplasm (Figure 5a). Type 2 and T3 mitochondria were located in clusters of
organelles throughout the ooplasm in the Stage III oocyte (Figure 5b). Type 2 mitochondria had transversely or randomly oriented cristae, often with enlarged intracristae spaces. By Stage III, the ultrastructure of T2 mitochon-
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dria was quite variable and included elongated rodshaped, branched, dumbbell, and donut configurations (Figure 6). Small, round inclusions were found in all types of mitochondria but were most prevalent in the T3 mitochondria. The two characteristics that differentiated Stage IV and V oocytes from Stage II and III oocytes were the absence of T3 mitochondria and the peripheral redistribution of the mitochondria in the Stage IV and V oocytes (Figures 7, 8). The latter thus formed a region referred to as the mitochondrial ring. The pleomorphic ultrastructural appearance of T2 mitochondria remained unchanged (Figures 7, 8, 10). Smooth Endoplasmic Reticulum. In Stage II oocytes, vesicular and more elongated cisterna of SER were primarily associated with the mitochondria in the Balbiani body (Figure 3). In Stage III oocytes, elongated SER was found aligned along the oolemma and nuclear envelop (Figures 5a,b). Also prevalent were vesicular SER, which were associated with mitochondria throughout the oocyte (Figure 6). In Stage IV and V oocytes, the vesicular SER was dispersed throughout the ooplasm (Figures 8, 10). Additional Ooplasmic Components. The ooplasm of Stage I oocytes contained multivesicular bodies (Figure 2) and macrobodies. Golgi were observed in the Balbiani body and in the cortical region in Stage II oocytes (Figure 9) and in the ooplasm of Stage III, IV, and V oocytes (Figures 5a, 7). Golgi subjacent to the oolemma were oriented with the maturing (concave) face in the direction of the granulosa cell layer. Macrobodies (Figure 3), which were scattered throughout the ooplasm, and microbodies were common in the
Stage II oocytes. Lipid droplets and a few yolk granules appeared to be more concentrated in the cortical region of the oocyte (Figure 9). Lipid droplets, macrobodies, and multivesicular bodies were observed throughout the Stage III oocytes (Figures 5a, b). Golgi were located within the clusters of mitochondria near the GV or in the cortical region with their maturing face oriented towards the center of the oocyte. In Stage IV oocytes, macrobodies had increased in number and multivesicular bodies appeared to decrease in number when compared to previous stages. The maturing face of the Golgi adjacent to the oolemma appeared to be randomly oriented. Vesicular SER was located adjacent to the nuclear envelop (Figure 8). In Stage V oocytes, yolk granules and macrobodies were scattered throughout the ooplasm (Figure 10). Many of the macrobodies possessed transosomes and had the appearance of yolk granules. Lipid droplets were concentrated in a concentric layer subjacent to the mitochondrial ring. At no stage in the development of the turkey primary oocyte was a yolk nucleus observed.
DISCUSSION This study defined the sequence of morphological and ultrastructural changes of primary oocytes in the slow growth phase of follicular development in mature turkey breeder hens. This description has not been reported previously and is important given the continued interest in elucidating both the physiological basis of follicular recruitment, particularly as the turkey ages (Hocking et al., 1988), and the relationship between
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FIGURE 1 (continued), d) A light micrograph of a Stage IV oocyte with lipid distributed in its cortical region. The Balbiani body is fully dispersed. (Bar = 67 pm). e) A light micrograph of a Stage V oocyte illustrating the zone-like distribution of organelles within the oocyte. Note the Stage I oocytes. (Bar = 108 /art).
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Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on March 15, 2015 FIGURE 2. A dense cluster of mitochondria (arrowheads and arrows) is an indication of the future Balbiani body in the Stage I oocyte. Large, ovoid, Type 1 mitochondria (arrows) and elongated Type 2 mitochondria (arrowheads) are apparent. Triangular-shaped intracristae spaces are also observed in some Type 2 mitochondria (arrow #2). Also observed are several multivesicular bodies (MVB) adjacent to the germinal vesicle (GV). (Bar = 1.4 ^m). FIGURE 3. In Stage II oocytes, mitochondria typically had a pleomorphic appearance. Type 2 mitochondria remained elongated but the orientation of their cristae was quite variable (arrowheads). Type 3 mitochondria, which were characterized by their filament-like cristae, were observed in Stage II oocytes (arrows; T3). Also note that the arrowheads are directed toward small dense inclusions in the mitochondrial matrix. A microbody (MB) and vesicular smooth endoplasmic reticulum (SER; thick arrow) and more elongated SER cisterna (arrow outline) are also observed. (Bar = 1.0 /mi). FIGURE 4. A cross section of a Type 3 mitochondrion (arrow) revealed the detailed configuration and filamentous appearance of its cristae. Type 2 mitochondria are also observed. (Bar = 2.4 /*m).
increased body weight and the subsequent decrease in egg production due to disruption to the normal follicular development (see Nestor et ah, 1980; Hocking, 1992; Renema et al., 1995). Whether this staging procedure is applicable to the growth and development of primary oocytes in immature turkey hens is not yet known.
In mature turkey hens, development of primary oocytes between 50 and 800 jiim in diameter can be divided into five stages based on its diameter, and the morphology, ultrastructure, and distribution of organelles and inclusions. Previously, staging of the avian primary oocyte from the pre-Balbiani stage to the stage characterized by zonation of the organelles and inclu-
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CARLSON ET Ah.
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FIGURE 5. a) In this Stage III oocyte, smooth endoplasmic reticulum (SER) (arrow) and a Type 3 (T3) mitochondrion (arrow 3) are adjacent to the nuclear envelop (arrowheads). Immediately above this Type 3 mitochondrion is a dumbbell shaped Type 2 mitochondrion. A Golgi body (G) and several lipid droplets (L) are also observed. (Bar = 2.4 ^m). b) A complex array of SER (arrow) is adjacent to the oolemma (arrowhead), which separates the oocyte from the granulosa cell layer (portions of granulosa cells are observed). Several elongated Type 2 (arrow 2) and Type 3 (arrow 3) mitochondria are also present in this cluster of organelles. (Bar = 2.4 fim).
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sions was based on a composite of several different species often of undocumented reproductive status. These species included the chicken (Gallus gallus), sparrow (Passer domesticus), pigeon (Columba livid), crow (Corvus splendens), myna (Acridotheres tristis), and the swallow (Hirundo rustica) or other feral species (see Romanoff, 1960; Guraya, 1989). Although fundamentally similar to other avian species, significant differences in the characteristics of the turkey primary oocytes compared to that of the primary oocytes of other species, particularly the chicken, were noted. Unlike in the chicken oocyte, lipid droplets did not aggregate around the GV opposite the Balbiani body in turkey oocytes. Furthermore, whereas lipid droplets were redistributed to the periphery in 170 to 260 /xm diameter oocytes in the chicken, a similar redistribution of lipid droplets in the turkey oocyte was not observed until Stage IV (351 to 500 [im in diameter). Golgi, which were a prominent feature in the early chicken oocyte (reviewed by Romanoff, 1960), were not a significant feature in the early primary oocyte of the turkey. Finally, a yolk-nucleus, which Guraya (1989) defined as
TURKEY PRIMARY OOCYTES
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FIGURE 6. Organelles in the dispersing Balbiani body of a Stage III oocyte include branching and dumbbell-shaped mitochondria (arrows). Macrobodies are evident (arrowheads) as is vesicular smooth endoplasmic reticulum (SER) (thick arrow) in the vicinity of the nuclear envelop (curved arrow). Variable amounts of vesicular SER were observed throughout the ooplasm in Stage II through Stage V oocytes. (Bar = 2.0 (im). FIGURE 7. Pleomorphic Type 2 mitochondria are in a more cortical position in the Stage V oocyte and form the mitochondrial ring. Granulosa cell extensions project into the ooplasm (arrowheads) and some appear to be enveloped by dilated vesicular smooth endoplasmic reticulum (black arrows). Golgi (white arrows) are also observed. (Bar = 2.5 /mi).
a component of the Balbiani body in the chicken oocyte involved in the multiplication of mitochondria, Golgi, and lipid droplets, was not observed in the turkey primary oocyte. It is suggested that the Stage I primary oocyte in the turkey was quiescent and that formation of the Balbiani body, which characterized the Stage II primary oocyte, was a manifestation of "follicular recruitment". This possibility is supported by Guraya's (1989) suggestion that the Balbiani body was the site of high metabolic activity, a prerequisite for further growth and development. The movement of organelles and inclusions during Balbiani body dispersal (Stage III) away from the more centrally located GV correlated with an increase in the growth of the Stage III oocyte relative to the GV. Such
observations suggest that the redistribution of organelles to the periphery of the primary oocyte has a functional role with regard to the transport of yolk precursor materials into the oocyte and possibly yolk granule formation. Likewise, the abundance of the pleomorphic T2 mitochondria, particularly the dumbbell configuration, is indicative of mitochondrial replication, a necessary prerequisite for oocyte growth and development. Although intramitochondrial yolk-crystals have been described in frog oocytes (Massover, 1971), the filamentous component of T3 mitochondria has not previously been described in either avian or amphibian oocytes (Schjeide et al., 1963a,b, 1966; Greenfield, 1966; Bellairs, 1967; Thiaw and Mattei, 1992; Kovacs et al, 1992). However, a filamentous component in mitochondria has been described in guinea pig oocytes (Anderson, 1974)
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if
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CARLSON ET Ah. *1
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on March 15, 2015 FIGURE 8. In the vicinity of the germinal vesicle in this Stage IV oocyte are elongated Type 2 mitochondria (arrows), macrobodies (arrowheads), and an abundance of vesicular smooth endoplasmic reticulum (thick arrows). The nuclear envelop (curved arrow) is observed. (Bar = 4.0 p.m). FIGURE 9. In this Stage II oocyte, the Golgi (white arrows), which are adjacent to the oolemma, are associated with vesicular smooth endoplasmic reticulum (thick arrow). Lipid droplets (arrow outlines) and yolk granules (double headed arrow) are also observed. Note the granulosa cells. (Bar = 2.2 iaa). FIGURE 10. Macrobodies (arrowheads) and yolk granules (YG) are observed in a Stage V oocyte. Some macrobodies have formed a dark central area (arrow) rendering them similar in appearance to the yolk granules. The vesicular smooth endoplasmic reticulum is dispersed throughout the ooplasm as are lipid droplets (arrow outline). (Bar = 2.8 jitm).
and neoplastic oncocytes (Tandler and Hoppel, 1972). In a review of oocyte ultrastructure, Anderson (1974) noted the paucity of SER in vertebrate oocytes. With its abundant vesicular SER, the turkey primary oocyte appears to be an exception. Likewise, rough endoplasmic reticulum, which was commonly observed in oocytes from other species (Anderson, 1974), was notably absent from the turkey's primary oocytes.
The close proximity of SER with the oolemma and Golgi in Stages II through Stage V suggests that the SER is involved in plasma membrane synthesis. It has been shown that SER is capable of synthesizing ceramides, which are transported to the Golgi to serve are a precursor for the synthesis of glycosphingolipids and sphingomyelins, both of which are components of the plasma membrane (Alberts et ah, 1989). Furthermore, in
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An understanding of the normal growth and development of the primary oocyte will contribute to our fundamental understanding of the oocyte maturation and the possible influence the primary oocyte plays in follicular recruitment and aberrant multiple hierarchy formation. In addition, the staging procedure described in this study will provide objective criteria defining the magnitude of maturation of isolated, in vitro matured turkey oocytes.
ACKNOWLEDGMENTS This paper is Scientific Article Number A7738; Contribution Number 9058 of the Maryland Agriculture Experiment Station (Department of Poultry Science). Research conducted by J. Carlson was in partial fulfillment of requirements for the Master's Degree.
REFERENCES Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts, and J. Watson, 1989. Molecular Biology of The Cell. Garland Publishing, Inc., New York, NY. Anderson, E., 1974. Comparative aspects of the ultrastructure of the female gamete. Pages 1-70 in: International Review of Cytology. G. H. Bourne and J. F. Danielli, ed. Academic Press, New York, NY. Bellairs, R., 1967. Aspects of the development of yolk spheres in the hen's oocyte, studied by electron microscopy. J. Embryol. Exp. Morphol. 17:267-281. Bubel, A., 1989. Microstructure and Function of Cells. Electron Micrographs of Cell Ultrastructure. Halsted Press, New York, NY. Carlson, J. L., 1993. Characteristics and morphology of developing yolkless oocytes in turkeys. Master's thesis. University of Maryland, College Park, MD. Chalana. R. K., and S. S. Guraya, 1979. Morphological and histochemical observations on the primordial and early growing oocytes of crow (Corvus splendens) and myna (Acridotheres tristis). Poultry Sci. 58:225-231. Gilbert, A. B., 1979. Female genital organs. Pages 237-360 in: Form and Function in Birds. A. S. King and J. McLelland, ed. Academic Press, London, UK. Greenfield, M. L., 1966. The oocytes of the domestic chicken shortly after hatching, studied by electron microscopy. J. Embryol. Exp. Morphol. 15:293-316. Guraya, S. S., 1989. Ovarian Follicles in Reptiles and Birds. Springer-Verlag, New York, NY. Hocking, P. M., 1992. Genetic and environmental control of ovarian function in turkeys at sexual maturity. Br. Poult. Sci. 33:437-448. Hocking, P. M., A. B. Gilbert, C. C. Whitehead, and M. A. Walker, 1988. Effects of age and early lighting on ovarian function in breeding turkeys. Br. Poult. Sci. 29:639-647. Kovacs, J., V. Forgo, and P. Peczely, 1992. The fine structure of the follicular cells in growing and atretic ovarian follicles of the domestic goose. Cell Tiss. Res. 267:561-569. Marza, V. D., and E. V. Marza, 1935. The formation of the hen's egg. Parts I-IV. Q. J. Microsc. Sci. 78:183-249. Massover, W. H., 1971. Intramitochondrial yolk crystals of frog oocytes II. Expulsion of intramitochondrial yolk crystals to form single membrane bound hexagonal crystalloids. J. Ultrastruct. Res. 36:603-620.
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pancreatic exocrine cells, the transfer of membranebound vesicles from the Golgi to the plasma membrane is thought to be responsible for the addition of surface plasma membrane during growth (Bubel, 1989), a mechanism that should be functioning in the growing oocyte. In Stage I oocytes, the Golgi had the maturing (concave) face oriented toward the GV. One of the functions of Golgi is synthesis of peptide signaling molecules (Alberts et al, 1989). Possibly in the Stage I oocytes, the Golgi may have been involved in synthesis of a signaling molecule that signaled the initiation of growth. In Stage II oocytes, larger, more well developed Golgi were found primarily in the cortical region of the oocyte and oriented with their maturing faces toward the granulosa cell layer. This change in orientation may reflect a plasma membrane synthesizing function in the immediate vicinity of the oolemma; however, in Stage IV and V oocytes, the orientation of the Golgi was more random. As noted above, the oolemma was becoming increasingly convoluted and the surface area is increasing, both of which not only necessitate the de novo synthesis of plasma membrane but an increase in the lipid and protein transport capacity across the oolemma. It may also reflect some activity of yolk granule formation. Macrobodies [membrane-bound clusters of transosomes surrounded by a common membrane (Greenfield, 1966)] and multivesicular bodies [membrane-bound vesicles that contain small vesicles, as defined by Bubel (1989)] play an important role in the initial development of certain types of yolk spheres (Greenfield, 1966; Bellairs, 1967; Paulson and Rosenberg, 1972; Schjeide et al, 1974). Although Paulson and Rosenberg (1972) indicated that yolk sphere formation began when the chicken primary oocyte was 2.5 to 3.0 mm in diameter, a few yolk granules were observed at all five stages of primary oocyte development in the turkey. An unexpected finding during LM examination of the primary oocytes was the difference in the number of primary oocytes having irregular GV between the Flocks A and B. All hens used for this study were Large White turkeys obtained from the same commercial source. However, the Flock A was a female line (selected for egg production) and Flock B was a male line (selected for high body weight) and consequently produce fewer eggs, possibly as a result of disruption in the development of the normal follicular hierarchy (see Nestor et al, 1980; Hocking, 1992; Renema et al, 1995). The male-line hens, which are selected for increased body weight and consequently suffer from reduced egg production, had a higher incidence of irregular GV than the oocytes of hens selected for egg production. If GV irregularity presages follicular atresia, it can be speculated that decreased egg production by Large White male-line hens may be due to a greater percentage of the primary oocytes undergoing follicular atresia than with hens genetically selected for egg production.
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CARLSON ET Ah. Origins and roles of mitochondria-like organelles. Growth 27:111-124. Schjeide, O. A., R. G. McCandless, and R. J. Munn, 1963b. Possible participation of RNA in formation of mitochondria-like organelles. Growth 27:125-128. Schjeide, O. A., R. J. Munn, R. G. McCandless, and R. Edwards, 1966. Unique organelles of avian oocytes. Growth 30: 471^89. Schjeide, O. A., L. Hanzely, S. J. Holshouser, and W. E. Briles, 1974. Production and fates of unique organelles (transosomes) in ovarian follicles of Gallus domesticus under various conditions. Cell Tiss. Res. 156:47-59. Sokal, R. R., and F. J. Rohlf, 1981. Biometry. W. H. Freeman and Co., New York, NY. Tandler, B., and C. L. Hoppel, 1972. Mitochondria. Academic Press, New York, NY. Thiaw, O. T., and X. Mattei, 1992. Natural degenerating mitochondria in ovarian follicles of a cyprinodontidae fish, Epiplatys spilargyreus (Teleost). Mol. Reprod. Dev. 32: 67-72. Yoshimura, Y., T. Okamoto, and T. Tamura, 1993. Ultrastructural changes of oocyte and follicular wall during oocyte maturation in the Japanese quail (Coturnix coturnix japonica). J. Reprod. Fertil. 97:189-196.
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Nestor, K. E., W. C. Bacon, and P. A. Renner, 1980. The influence of genetic changes in total egg production, clutch length, broodiness, and body weight on ovarian follicular development in turkeys. Poultry Sci. 59:1694-1699. Paulson, J. L., and M. D. Rosenberg, 1972. The function and transposition of lining bodies in developing avian oocytes. J. Ultrastructure Res. 40:25-43. Perry, M. M., A. B. Gilbert, and A. J. Evans, 1978a. The structure of the germinal disc region of the hen's ovarian follicle during the rapid growth phase. J. Anat. 127: 379-392. Perry, M. M., A. B. Gilbert, and A. J. Evans, 1978b. Electron microscope observations on the ovarian follicle of the domestic fowl during the rapid growth phase. J. Anat. 125: 481-497. Renema, R. A., F. E. Robinson, V. L. Melnychuk, R. T. Hardin, L. G. Bagley, D. A. Emmerson, and J. R. Blackman, 1995. The use of feed restriction for improving reproductive traits in male-line large white turkey hens. 2. Ovary morphology and laying traits. Poultry Sci. 74:102-120. Romanoff, A. L., 1960. The Avian Embryo. Macmillan, New York, NY. Schjeide, O. A., R. G. McCandless, and R. J. Munn, 1963a. Further observations on the developing avian oocyte.