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Cite this page: Hill, M.A. (2020, June 3) Embryology Ferret Development. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Talk:Ferret_Development
Oogenesis and the development of the ovarian interstitial tissue in the ferret
By Ruth Deanesly
A.R.C. Institute of Animal Physiology, Babraham, Cambridge
(Received 2 May 1969) J. Anat. (1970), 107, 1, pp. 165-178 165
With 22 figures
Odégenesis and the meiotic prophase occurring in foetal life have been described in the mouse (Brambell, 1927; Borum, 1961), the rat (Beaumont & Mandl, 1962), the human and macaque (Baker, 1963; Van Wagenen & Simpson, 1965), the pig (Black & Erickson, 1968) and other species. In the rabbit, however, Teplitz & Ohno (1963) reported that the meiotic prophase was postnatal and this was further studied by Peters, Levy & Crone (1965). More recently, postnatal odgenesis has been described in the hamster (Weakley, 1967; Greenwald & Pepler, 1968), but, hitherto, no information has been available for the ferret, in which ovo-implantation occurs on day 12 or 13 and pregnancy lasts 6 weeks, (41-44 d: J. Hammond, personal communication). In the present paper the development of the ovary is described from the 32nd d of pregnancy in the ferret to 35 d post partum (p.p.) a period which includes not only odgenesis and the meiotic prophase which is postnatal, but also the development of primordial follicles and the differentiation of the characteristic lipid-containing interstitial cells.
MATERIALS AND METHODS
Dated embryos and neonatal ferrets were obtained from the colony in the School of Agriculture, Cambridge. The ovaries were fixed in Susa, Bouin or Ciaccio fluids. The last method, for preserving lipids, consists in fixation in freshly made up 5% potassium bichromate 8 ml, 40% formalin 2 ml and glacial acetic acid 0-05 ml for 48 h, followed by postchroming for 3 d in 3% potassium bichromate, washing in running water 24 h, dehydration and embedding. Sections were counter-stained in oil red O or Scharlach R to show cell lipids, which have the same distribution as those in similar frozen sections. Commonly one ovary was fixed for lipids and the other for study of micro-anatomical detail. Abundant material was available to study the course of the meiotic prophase but for reasons given on p. 175 no attempt was made to count nuclei in different stages.
DEVELOPMENT OF THE OVARY
31 to 39 d embryos. Ovaries from 19 embryos in this group were examined; stages were available for each day from d 34. At 31-32 d the ovaries were still very small and broadly attached by stromal tissue to the mesonephros; two entire embryos were sectioned. Cortex and medulla were distinguishable in these early ovaries; o6gonia, some in mitosis, were mainly in the cortex but others singly and then in groups of two to six also appeared in the medullary stroma, where they had apparently migrated as proliferation took place. Odgonial nuclei varied in diameter from 7 to 14 um. Typically each had a single nucleolus. The ovaries grew steadily during this period but showed no conspicuous histological changes. Stromal cells with oval, pear-shaped or ‘triangular’ nuclei ramify among the odgonia (Figs. 1, 3); as this tissue increases it tends to divide the odgonia into groups, a condition accentuated in later stages (Figs. 2, 6).
During the last 2 weeks of foetal life the ovaries continue to increase in size and odgonia multiply by mitosis. In the seven embryos removed shortly before birth (37-39 d), there were no striking changes and the odgonia did not pass into the meiotic prophase.
Foetal and neonatal ferrets ; 40-46 d p.p.
This group contained 12 ferrets of which seven were postnatal. Mitotic figures were seen in ovarian stroma and oégonia (Fig. 4), but except in their increased size the ovaries hardly differed from those already described. The cortex, bounded by coelomic (‘germinal’) epithelium formed a large proportion of the total ovarian volume. Both cortical and medullary odégonia had vesicular nuclei of varying size (7-11 wm diameter) and with one or more distinct nucleoli. The earliest rete tubules were forming in the medulla 1 day after birth.
Neonatal ferrets 6-16 d old; the meiotic prophase and interstitial cell differentiation
Ovaries from 15 females of this group were examined. The youngest ferret in which meiosis had definitely begun was 6 d old. So far only a small proportion of odgonia was affected, up to a dozen in a (horizontal) section, but leptotene, zygotene and pachytene stages could be distinguished, of which pachytene were the most common. Some oégonia were in mitosis.
In a smaller, 7 d ovary no meiotic stages could be seen, but at 8 and 9d meiosis was in progress in most odcytes in the inner half of the cortex (Fig. 5). The proportion in which meiosis has begun varies in the three 8 and 9d females studied and is rather lower in one of the 9 d animals. Figs. 6-8 from a 10d ovary show nuclei in zygotene, pachytene (early and late), and early diplotene; among them lie the nuclei of the stroma cells. There are also numerous dense pachytene nuclei, singly and in groups, some of which may be degenerating, like the odcytes with dense chromatin and eosinophil cytoplasm in the rat, described as Z cells by Beaumont & Mandl
Development of the ovary in the prenatal and early postnatal ferret. Sections through ovaries from 37 d foetus to neonatal young up to 28 d p.p.
Fig. 1. No. 23, 37d foetus (x 135). Most of the odgonia are in the cortex, but some in the medulla.
Fig. 2. No. 14, 1 d p.p. (44 d pregnancy) ( x 110). This ovary is larger than the previous one and o6gonia have increased in number; many can be seen in the medulla.
Fig. 3. No. 23, 37d p.p. (x 800). Resting odgonia interspersed with stromal fibrous cells. Fig. 4. No. 5, 2d p.p. (x 800). Odgonia of varying size, some in mitosis, and stromal cells.
(1962). Such Z cells are not common at 10d p.p. in the ferret. Groups of odgonia at the outer edge of the cortex, as in the 9 d ovary, are still not involved in meiosis (Figs. 5, 6) but later, from about 14 d onwards, these gradually undergo the meiotic changes. Nuclei in diplotene can be found at 10 and 14d, but they are scarce.
At 13-14 d the predominant meiotic stage is pachytene (Fig. 10) occurring in both cortical and medullary oocytes, but other stages are also present. One 14d ferret ovary shows eggs degenerating in groups and an increase in dark staining fibrous connective tissue in the cortex, leading to a disorganized appearance in certain areas (Fig. 9).
In two 15d ferrets the ovaries again show all stages of meiosis including many pachytene nuclei, but resting odgonia are still present at the edge. In the ovarian cortex there is connective tissue proliferation and groups of degenerating odcytes similar to those in the 14d ovary, but the degeneration process is less advanced.
A few primordial follicles can be distinguished next to the medulla at 16d, flattened stroma cells becoming the encircling pregranulosa cells (cf. Fig. 20). These are cells of the kind which have ramified among the odgonia from the earliest stages of ovarian development. There is no reservoir of distinguishable epithelial cells to enclose the odcytes of the primordial follicles. At 16d cell boundaries are disappearing from whole groups of degenerating odcytes in some parts of the cortex.
From d 7, the precursors of the interstitial tissue, a few lipid-containing cells originating in the medullary stroma and varying in size and shape, can be seen. During the next week they increase and differentiate into epithelioid, lipid-containing cells with rounded nuclei. They form glandular lobules first distinguishable in the broad hilar region away from the odcytes, and gradually spread round the medulla. The cells enlarge (Figs. 11, 12) but their size varies in different parts of the ovary during development. By d 16, lipid-containing cells are common but most of the medulla still remains undifferentiated.
In sections from 14 to 16d ovaries, stained for lipid, the oGcytes, regardless of their nuclear condition, also show lipid granules, though they are paler than those of the interstitial cells. Cytoplasmic lipid also occurs in later odcytes, as found by Mainland (1931) who studied ferret eggs at about the time of ovulation.
In this group of ovaries, small rete tubules appear in the medulla, many of them close to the odcytes (Fig. 10). The cells lining these tubules contain no lipid and the long axes of their oval nuclei are perpendicular to the lumen. Early development of the ovarian rete has been described by Kingsbury (1913) in the cat, by Torrey (1947) in the rat, by Sauramo (19545) in the human, and others. Torrey (1947) concludes that the rete develops in situ from the gonad rudiment, not from the mesonephros.
Fig. 5. No. 130, 9d p.p. (x 275). Part of the ovarian cortex, showing the onset of the meiotic phase in the inner groups of odcytes. The nuclei here are mainly pachytene. Outer odgonia are still resting or dividing normally.
Fig. 6. No. 131, 10d p.p. (x 275). Similar to Fig. 5 but more odcytes are in meiotic stages. The connective tissue is prominent.
Fig. 7, 8. No. 131, 10 d p.p. (x 1180). Odcytes showing leptotene, zygotene and pachytene stages; among them can be seen the ‘triangular’ or oval nuclei of stromal cells.
In the ferret, rete tubules appear to take up degenerating odcytes (Figs. 10, 19) which can be seen in the lumen as early as d 16. The characteristic rete epithelium differs from that of the earliest follicles (Fig. 22).
Ferrets 17-24 d old: follicle formation, reduction of odcytes and interstitial cell proliferation
Ovaries from 15 females were examined during this latter part of the meiotic phase. Many odcytes had degenerated, but only in one female was the cortical area substantially reduced and that at 23 d p.p. The degeneration process is gradual in the ferret and is not synchronized over different areas of the same ovary. Zygotene, pachytene and diplotene stages are all present (Figs. 6, 7, 13, 14) and it appears that oGdcytes can finally degenerate at any stage of meiosis and not necessarily after the condensed pachytene stage. Condensed pachytene nuclei, though common, show no consistent increase in numbers during this week. Figs. 15, 16 (24 d p.p.), illustrate areas of odcyte degeneration becoming occupied by stroma cells. A larger area is seen in Fig. 9 where the dark-staining connective tissue is prominent in the cortex, whence many odcytes have disappeared. A similar proliferation of connective tissue appears to take place in human and macaque ovaries during odcyte degeneration (Van Wagenen & Simpson, 1965).
During this period, odcytes in early and late diplotene become more numerous, especially at the inner edge of the cortex. Most odcytes in the medulla degenerate. Primordial follicles increase and, in a few, the epithelium has become cuboid and surrounded by a connective tissue sheath. The transition between flattened and cuboid, pregranulosa cells can be seen in Figs. 21, 22.
Interstitial tissue increases throughout the ovary and by 17d p.p. may reach the inner edge of the cortex (cf. Fig. 11). At 19d p.p. the cells vary in size, the larger ones being seen in the hilar region (Figs. 17, 18), and at 22 d it is the cells differentiating in the medulla, probably the most recent, which are smallest. At that time a good deal of undifferentiated stromal tissue persists in the medulla, but gradually the whole area is taken over by lipid-containing interstitial tissue with its accompanying blood vessels. Rete tubules are still present and in the lumen degenerating odcytes can frequently be seen (Fig. 19).
Fig. 9. No. 121, 14d p.p. (x 110). Low power view to show disorganization of the ovarian cortex and conspicuous proliferation of stromal tissue during odcyte degeneration, more advanced in this ovary than in some later ones.
Fig. 10. No. 121, 14d p.p. (x 275). Odcytes in meiosis, some degenerating. Odcytes, in condensed pachytene appear to be passing into the prominent rete tubule (arrowed), on the right.
Fig. 11. No. 143, 19d p.p. (x 275). Many odcytes have disappeared from this part of the cortex leaving vacuolated areas; a primordial follicle can be seen on the right at the junction of cortex and medulla, and near the left, a rete tubule cut transversely. Adjacent to the cortex are lobules of differentiating interstitial cells.
Fig. 12. No. 11, 28d p.p. (x 275). In this ovary the cortex is reduced in depth and consists mainly of primordial follicles. A primary follicle with cuboid epithelium lies near the glandular interstitial cells of the medulla.
Fig. 13. No. 144, 18 d p.p. (x 800). Odcytes in zygotene, pachytene and early and late diplotene.
Fig. 14. No. 147, 21 d p.p. (x 800). Area including degenerating odcytes, zygotene, pachytene, condensed pachytene and diplotene stages.
Fig. 15-16. No. 137, 24 d p.p. (x 800). Vacuolated areas in the cortex, and odcytes in diplotene becoming encircled by stromal cells, the precursors of the follicular epithelium. Early development of ferret ovary 173
Ferrets 25-35 d p.p.
The ovaries of six females were examined. This is the period when the cortex contracts as the degenerating odcytes are gradually absorbed. All that remains by d 28 is a small ‘cortical’ area consisting mainly of primordial follicles (Fig. 12) overlying but not completely covering a much larger area of interstitial and stromal cells. As late as d 26, however, pachytene, degenerating pachytene and early diplotene stages are not uncommon, and some persist at d 35. Pregranulosa cells are now cuboid in more of the primordial follicles (Fig. 22), some of which have acquired a fibrous sheath.
At 35 d characteristic groups of larger rete tubules can be seen near the hilus and their appearance is similar to that of the tubules first seen at 1 d p.p. At 27 and 28 d, tubules near the residual ovarian cortex contain odcyte debris as do some of the medullary tubules.
The meiotic phase of the odcytes and the differentiation of the glandular interstitial cells in the ferret ovary both take place in the first month of postnatal life and can be well studied in serial sections. During the last 10 d of pregnancy the growing foetal ovary consists of a cortex of odgonia, some in mitosis, bounded by coelomic epithelium and a medulla, mainly of stromal tissue, in which lie small groups of additional odgonia. Fibrous stromal tissue also ramifies among the cortical o6gonia dividing them into groups.
Od6gonia in the present series did not pass into the meiotic phase before 6 or 10d p.p., and od6gonia at the outer edge of the cortex were not at first involved. By d 14, however, such resting o6gonia, though still present, were much less common; odcytes in leptotene, zygotene, pachytene and diplotene were all seen and these different prophase stages continued to be present up to d 22 p.p. Pachytene appeared to be the longer lasting and most common stage, as other observers have noted. This included the ‘bouquet’ type of nucleus and the type in which the chromatin is very condensed and almost pycnotic, a few of them similar to the ‘Z’ cells of the rat ovary. Some of these oGcytes are almost certainly degenerating, but many, especially towards the end of the meiotic prophase, seem also to degenerate during other prophase stages.
Most of the odcytes in the medulla pass into meiosis concurrently with those in the cortex but then degenerate, though a few reach the dictyate stage and may be enclosed as primordial follicles.
The ovarian cortex of the ferret during meiosis resembles that of the macaque and the human (Van Wagenen & Simpson, 1965, Pl. 33-36 and 12-14). In the 14-21 day ferret, as in the Primates, the connective tissue is very prominent among the odcytes and stains deeply (Figs. 10, 17) especially in regions of odcyte degeneration.
Fig. 17. No. 143, 19 d p.p. (x 47). Section through ovary stained to show distribution of lipidcontaining interstitial cells. Some of these can be seen next the cortex (Fig. 11) but they are larger and more abundant at the opposite end of the ovary.
Fig. 18. No. 143, 19 d p.p. (x 800). Lipid-containing interstitial cells at a higher magnification.
Fig. 19. No. 144, 18 d p.p. (x 800). Oblique section through a rete tubule containing degenerating oocytes.
Examination of serial sections made it obvious that to determine the proportions of leptotene, zygotene, pachytene and diplotene stages and their transitional forms on any given day with a reasonable degree of accuracy would involve a wholly disproportionate amount of work without providing significant results. Borum (1961) in her paper on meiosis in the mouse, states that her quantitative data should be considered as a ‘mere indication only’ for several reasons, including difficulties of classifying the stage of prophase owing to transitional stages and variations in the rate of ovarian development associated with litter size. In the ferret, the high proportion of degenerating odcytes must also be considered. Odcytes which have lost their cell boundaries and show aberrant nuclear formations commonly degenerate in groups. Although this degeneration has begun by d 14, the process continues till d 24 or later; as late as d 23 p.p. in at least one animal the total area of the cortex did not seem substantially reduced. Later, however, by d 25—28, most of the original cortex has apparently disappeared and the surviving odcytes, many enclosed in primordial follicles, occupy a small area on the surface of the ovary, which now consists mainly of glandular interstitial and vascular tissue. The smallness of the odcyte areas in sections through the 4-5-week-old ferret ovary indicates that a high proportion of the original odcytes have disappeared during meiosis.
Primordial follicles are first seen at the medullary edge of the cortex about d 16 p.p. The surrounding flattened pregranulosa cells derive from the stroma which has ramified among the odcytes from early stages of ovarian development (Figs. 3, 4, 7, 20). Their gradual transition to cuboid epithelial-type cells can be traced on d 20-28 (Figs. 20-22). In older embryological studies, such as that of Brambell (1927) on the mouse, and more recently Van Wagenen & Simpson (1965) on the human and macaque, an epithelial origin is attributed to the granulosa, from cells below the coelomic epithelium or interspersed among the odcytes. The evidence for a distinct epithelial origin of the pregranulosa cells however, is not strong. Sauramo (1954a) who also studied the human embryonic ovary states that ‘the cells of the follicle epithelium quite obviously develop from stroma cells’. Peters et al (1965; Fig. 7) show early primordial follicles in a 21 d rabbit, where the flattened cells round the follicles are similar to those of the adjacent strands of stromal tissue. In a later paper, seen after completion of the present work, Peters & Pedersen (1967) have described the origin of follicular epithelial cells from similar stroma cells in the early mouse ovary; the cells were traced by injection of tritiated thymidine.
The glandular interstitial cells which occupy most of the ferret ovary at 3-4 weeks old, are similar to those in the young adult. They derive from the stroma independently of cortical or medullary odcytes, when only primordial follicles have developed ; they proliferate rapidly during postmeiotic degeneration. Their differentiation
Fig. 20. No. 134, 22d p.p. (x 500). In the inner part of the cortex, on the right, odcytes in diplotene are enclosed by stromal cells to form follicles.
Fig. 21. No. 143, 19d p.p. (x 800). Above, early follicle formation, below a vacuolated area whence degenerate odcytes have disappeared.
Fig. 22. No. 11, 28 d p.p. (x 800). Primordial follicles showing transition from flattened to cuboid epithelium. On the right a transverse section through a rete tubule. RUTH DEANESLY Early development of ferret ovary 177
and increase in the 16-28 d ferret suggest a response to a specific pituitary gonadotrophic discharge.
In the foetal ferret testis the lipid-containing interstitial tissue appears well differentiated by the 31st d of pregnancy. This earlier development, as compared with that of the female, has been observed in other species such as cattle (Bascom, 1923).
In the ferret, which has a 42 d pregnancy, the meiotic phase of the odcytes and the differentiation of the glandular interstitial tissue take place over about the same postnatal period. The ovaries of 24 foetal and 43 early postnatal animals have been examined in serial sections from the 32nd d of pregnancy to 35 d after birth.
Active meiosis occurred between 10 and 21 d p.p. and degeneration and absorption of many odcytes from d 14 onwards; the process is gradual and odcytes do not all pass into the meiotic phase together within an ovary. At the end of the meiotic phase the cortex of the ovary is much reduced; probably 60-80% of the original odcytes have disappeared. Degenerating odcytes can be seen in the lumen of rete tubules which originate in the ovarian medulla and are well developed in the postnatal ovary. Primordial follicles with flattened surrounding cells can be found by day 16 p.p. These pregranulosa cells derive from the fibrous stromal tissue ramifying among the odcytes and, in earlier stages, among the oégonia.
Lipid-containing interstitial cells begin to differentiate from medullary stroma cells about d 14 and shortly afterwards they form glandular lobules of epithelioid cells at first most abundant in the broad hilar region. By d 21 and later, typical glandular interstitial tissue occupies most of the ovary.
The author is much indebted to Mr J. Hammond, School of Agriculture, Cambridge, who generously supplied and fixed the dated ovaries for this work from his ferret colony.
A grant from the Medical Research Council and the hospitality of the Agricultural Research Council are gratefully acknowledged.
The microphotographs are the work of Mr Gallop.
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