Cell Tiss. Res. 184, 143 154(1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Ultrastructure of Rat Ovarian Interstitial Gland Cells during Pregnancy Irvin E. Lawrence, Jr., Hubert W. Burden, and Marilyn L. Capps Department of Anatomy, School of Medicine, East Carolina University, Greenville, North Carolina, USA
Summary. The fine structure of the interstitial gland of the rat ovary was studied at estrus and on Days 4, 6, I0, 14 and 18 of pregnancy. At estrus, ovarian interstitial cells have small nuclei with dense irregular clumps of heterochromatin. Mitochondria are small and rod-shaped and have predominantely lamellar cristae. Numerous osmiophilic lipid droplets are present. At Days 4 and 6, nuclear heterochromatin is reduced, and nucleoli are larger and complex. Mitochondria are enlarged and often bizarre-shaped and have tubular cristae. Golgi and smooth endoplasmic reticulum are more conspicuous. At Day 10, prominent ultrastructural features include nuclei with conspicuous heterochromatin, smaller mitochondria with both lamellar and tubular cristae, numerous ribosomes and lipid droplets with decreased osmiophilia. At Days 14 and 18, nuclei have increased heterochromatin, mitochondria are small and have lamellated cristae and an increase in the size and number of lipid droplets occurs. These observations suggest that steroidogenic activity of interstitial cells is highest during the first half of pregnancy and regresses during the last half. It is suggested that the interstitial gland is an important ovarian source of pregnancy hormone(s) during the first half of gestation and that L H may modulate steroidogenic activity in this ovarian component. Key words: Ovary (Rat) - Pregnancy - Interstitial gland - Ultrastructure.
Introduction The interstitial gland of the mammalian ovary possesses ultrastructural, histochemical and biochemical features of steroid-secreting cells (Guraya, 1973; Mossman and Duke, 1973a, b). Previous electron microscopic studies have demonstrated that this ovarian component responds to endogenous and exogenous gonadotrophins (Carithers and Green, 1972 a, b; Carithers, 1976). An adrenergic Irvin E. Lawrence,Jr., Department of Anatomy, School of Medicine, East Carolina University,Greenville,North Carolina 27834, USA
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i n n e r v a t i o n o f the interstitial g l a n d has been d e m o n s t r a t e d b y several investigators (Unsicker, 1970; Burden, 1972; Unsicker, 1974; Svensson et al., 1975; L a w r e n c e a n d Burden, 1976), a n d it has been suggested t h a t in a d d i t i o n to h y p o p h y s e a l influences, steroidogenic activity o f the gland m a y also be affected by local n e u r a l stimuli (Svensson et al., 1975; L a w r e n c e a n d Burden, 1976). T h e p u r p o s e o f the present s t u d y was to d e m o n s t r a t e at the fine structural level the m o r p h o l o g y o f interstitial g l a n d cells d u r i n g pregnancy. These o b s e r v a t i o n s also f o r m a basis for studies o f the response o f interstitial cells to d e n e r v a t i o n d u r i n g pregnancy, to be p u b l i s h e d later.
Materials and Methods Animals. Female nulliparous Sprague-Dawley rats weighing 165 to 220 g were housed in a room with a 14-h light (0500 to 1900 h) and 10-h dark regimen. Estrous cycles were monitored daily between 0800 h and 1000 h by microscopic evaluation of aqueous vaginal lavages. To establish pregnancy, females in proestrus were housed with a male of proven fertility. The day on which spermatozoa were found in the vaginal lavage was designated as Day 1 of pregnancy. Rats in estrus and on Days 4, 6, 10, 14 and 18 of pregnancy were used in this study. ElectronMicroscopy. For fixation, a thoracotomy was performed and the trocar was placed through the left ventricle and into the ascending aorta. The animal was perfused preliminarily with Earle's balanced salt solution. After a briefwash with the salt solution, the animal was perfused with 3 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). The ovaries were dissected, minced and immersed in fixative to achieve a total fixation time of one and one-half to two hours. After a wash in phosphate buffer the tissue was post-fixed in 1% osmium tetraoxide, stained en bloc with uranyl acetate, (Karnovsky, 1967) dehydrated rapidly, and embedded in Maraglas. Thin sections were cut on a Porter-Blum MT 2-B ultramicrotome and stained with 2 % uranyl acetate and lead citrate (Reynolds, 1963). The tissues were viewed with a Philips EM 201.
Results Estrus. Nuclei in interstitial cells o f estrus rats are small a n d often deeply indented. Dense irregular c l u m p s o f h e t e r o c h r o m a t i n line the inner nuclear m e m b r a n e . A single nucleolus is often eccentrically l o c a t e d a d j a c e n t to h e t e r o c h r o matin. N u c l e o l o n e m a a n d p a r s a m o r p h a are present. M i t o c h o n d r i a are small a n d rod-shaped. Some cristae are tubular, b u t m o s t are l a m e l l a r (Fig. 1). The m i t o c h o n d r i a are frequently l o c a t e d in the cell periphery. T h e G o l g i c o m p l e x is c o m p o s e d o f a parallel stack o f saccules with a few associated vesicles. S m o o t h e n d o p l a s m i c reticulum is n o t conspicuous. A few scattered r i b o s o m e s a n d lysosomes are present. L i p i d d r o p l e t s are n u m e r o u s a n d a p p e a r p o l a r i z e d in the cell, occurring n e a r the c a p i l l a r y surface o r a l o n g the b o r d e r a d j o i n i n g connective tissue. M o s t o f the d r o p l e t s are u n i f o r m l y osmiophilic, a l t h o u g h s o m e d r o p l e t s within a single cell m a y be c o m p l e t e l y leached. Intercellular space is usually filled with a flocculent material. Day 4. Cellular a n d n u c l e a r h y p e r t r o p h y are m o s t a p p a r e n t in this group. Nuclei are enlarged a n d spherical, r o u n d to oval in outline, a n d the h e t e r o c h r o m a t i n is reduced. The nucleoli have a s s u m e d a m o r e central p o s i t i o n a n d are larger a n d m o r e
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Fig. 1. Ovarian interstitial cells from an estrus rat. Mitochondria (M) are small and have lamellar cristae. Lipid droplets (L) of varying sizes are present. Aggregates of nuclear heterochromatin (H) are present. The plasmalemmata of two adjacent cells form a tight junction (arrow). • 17,500
complex. The mitochondria are enlarged, and packed with tubular cristae. Some mitochondria are attenuated, and give the appearance o f division (Fig. 2). Other mitochondria are cup-shaped or annular. Sometimes mitochondria are closely associated with lipid droplets and/or smooth endoplasmic reticulum. Some mitochondria appear to be pinching off cytoplasm (Fig. 4) or "engulfing" other mitochondria (Fig. 5). The Golgi complex is larger and often has dilated cisternae. A few polysomes and scattered ribosomes are present. Distinct profiles of smooth endoplasmic reticulum are difficult to recognize. Filopodia of the steroidogenic cells interdigitate within the intercellular space. Nexuses are seen between some of the abutting cellular processes (Fig. 3).
Day 6. The nuclei are similar to Day 4, however, the heterochromatin is reduced. Nucleoli are less frequently seen. Mitochondria are similar in size and number to Day 4, but the number of cristae is reduced and some cristae are now lamellar. Mitochondria are often contiguous to lipid droplets. Lipid droplets are more uniformly osmiophilic. An extensive smooth endoplasmic reticulum occurs and vesicles o f the endoplasmic reticulum surround some of the lipid droplets (Fig. 5).
Fig. 2. A portion of an ovarian interstitial cell from Day 4 of pregnancy. The nucleus is more vesicular, heterochromatin (H) is reduced and a well organized nucleolus (N) is present. Golgi (G) apparatus is enlarged with stacks of saccules and vesicles. Mitochondria (34) are enlarged, have more tubular cristae, may be closely associated with lipid (L) droplets, and often assume complex shapes, x 16,625 Fig. 3. An extensive gap junction (arrows) between the plasmalemmata of two interstitial ceUs. Day 4 of pregnancy. Mitochondrion (34) with tubular cristae. • 23,750
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Fig. 4. Ovarian interstitial cells from Day 6 of pregnancy. An elongate mitochondrion (h4) appears to be in the process of"pinching off' cytoplasm. Other mitochondria appear annular(AM). At this time, there is a close association between mitochondria, smooth endoplasmic reticulum (arrows) and lipid droplets (L). Filopodia (F) enclose intercellular space. • 16,625
Fig. 5. Cytoplasmic features of ovarian interstitial cells from Day 6 of pregnancy. Note the numerous Golgi (G) profiles, extensive smooth endoplasmic reticulum (arrows) often in close association with lipid (L) droplets. Mitochondria are complex. Some appear to be in the process of "engulfing" other mitochondria (1), while other mitochondria (2) appear to have already been "engulfed". • 16,625 Fig.6. Interstitialcell, Day 6 ofpregnancy.Acilium(C) projects into the intercellular space(IC), x 16,625
Fig. 7. Portions of interstitial cells, Day 10 of pregnancy. Mitochondria (M) are rod-shaped and have a dense matrix. Polysomes (P) are present and lipid droplets (L) have a homogenous, slightly osmiophilic matrix. • 23,750 Fig. 8. Cytoplasmic features of interstitial cells, Day 14 of pregnancy. The contents of the lipid droplets (L) are partially leached. Mitochondria (M) are simple rods with both lamellar and tubular cristae. A tight junction (arrow) is present between adjacent cells. • 23,750
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Fig. 9. Portions of interstitial cells, Day 18 of pregnancy. The nucleus is irregular and heterochromatin (/4) is clumped. Lipid droplets (L) are large and mitochondria (M) are small and have lamellar cristae. • 17,500
Sometimes annular mitochondria may surround the endoplasmic reticulum-lipid droplet complex (Figs. 2, 4, 5). Golgi cisternae and vesicles are often associated with the smooth endoplasmic reticulum-lipid droplet complex. Numerous Golgi profiles occur throughout the cytoplasm (Fig. 5). Cilia often project into the intercellular space (Fig. 6). The basal body of these cilia is always within the vicinity of the Golgi complex.
Day 10. The nuclei are large and mostly spherical. The heterochromatin is conspicuous along the nuclear perimeter. A single nucleolus occurs in most cells. Mitochondria are usually small and rod-shaped and frequently located along one edge of the nucleus. Cristae are both tubular and lamellar, and occasionally dense granules occur in the matrix. The Golgi complex is prominent with lamelliform components and associated vesicles. The smooth endoplasmic reticulum is poorly visualized. Many free ribosomes occur. Lipid droplets are less osmiophilic than Day 6 (Fig. 7). Lipid droplets often occur in one pole of the cell near the capillary and/or connective tissue space. The intercellular space is pronounced. The space appears to be empty, although occasional membranous profiles may be present. Coated vesicles in the steroidogenic cells occur along the intercellular space. Cilia that are sometimes present in the vicinity of the Golgi complex extend into the intercellular space. Day 14. Nuclei of many Day 14 interstitial cells are indented. Some nuclei are large and spherical. The heterochromatin is more dense along the inner nuclear
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membrane. The nucleoli are conspicuous and have a distinct nucleolonema and pars amorpha. Mitochondria are small and have lamellated cristae (Fig. 8). Dense granules seldom occur in the mitochondrial matrix. In some cell, lipid droplets are few and the lipid has an electron-lucent center (Fig. 8). There is little smooth endoplasmic reticulum. Polyribosomes are present. On Day 14 there is less intercellular space. Filopodia are more elongate and interdigitated. Some tight junctions exist.
Day 18. Nuclei are smaller, oval to irregular in shape, indented and have dense heterochromatin. The nucleoli are infrequent. Mitochondria are small and have lamellated cristae (Fig. 9). There appear to be few electron dense granules in the mitochondrial matrix. The mitochondria are more random in distribution, often dispersed between lipid droplets. An increase in the number and size of lipid droplets occurs. The lipid is often partially leached. There are free ribosomes, with scant rough endoplasmic reticulum. The Golgi complex is small.
Discussion
Although the interstitial gland has been recognized in the mammalian ovary for over a century (Pfliiger, 1863) the significance and importance of this structure has been largely overlooked by both anatomists and physiologists. In our laboratory we have directed our attention to studies of the interstitial gland during pregnancy (Lawrence and Burden, 1976; Burden and Lawrence, 1977). The present study has demonstrated changes in the interstitial gland during pregnancy and correlates well with our histochemical observations of A S-3fl-hydroxysteroid dehydrogenase (3 flHSD) activity in the rat ovary during pregnancy. The enzyme activity (with pregnenolone substrate), which is interpreted as an index of the capacity of tissue to secrete progesterone, was most pronounced in the interstitial gland through Day 10 (Burden and Lawrence, 1977). By Day 14, activity in the interstitial gland and corpus luteum was similar and by Day 18, the site of greatest 3fl-HSD activity was the corpus luteum (Burden and Lawrence, 1977). Thus at Day 18, the interstitial gland has less capacity to secrete progesterone as determined by 3 fl-HSD activity and the fine structure of the cells suggests decreased steroidogenic activity. The fine structural changes in interstitial gland cells observed during pregnancy are both quantitative and qualitative. Nuclear and cytoplasmic hypertrophy characterize the first half of pregnancy, whereas during the last half of pregnancy, morphological features suggest a functional regression of interstitial gland cells. Contrary to the conclusion of Guraya (1972), qualitative changes appear in mitochondrial granules, the lipid droplets and the intercellular substance. Quantitative changes were characteristic of filopodia, nexuses, and cilia. Carithers and Green (1972a) observed nuclear and cytoplasmic regression following hypophysectomy. Then in sequal, they carefully monitored the stimulatory effects of gonadotrophin on these structures (Carithers and Green, 1972b). These observations led Carithers to conclude that LH is primarily cytoplasmic in its effect (Carithers, 1976). The earliest obvious change was in hypertrophy of the mitochondria. This was succeeded by other cytoplasmic changes
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leading to membranogenesis that was not completed until after the nuclear changes had been completed (Carithers, 1976). Initiation and development of the nuclear changes were prolonged. Interstitial cells during the first half of pregnancy were highly stimulated. In comparison to estrus, the nucleus and nucleolus were greatly enlarged and vesicular. At Day 4, a spherical nucleus, characteristic of active steroid cells, was present. A larger nucleus appeared in Day 6 interstitial cells. A prominent nucleonema and fibrillar substance constituted the nucleoli at this time. And thinly dispersed, peripherally disposed heterochromatin was present through Days 4 and 6 and extended into Day 10. These nuclear changes are suggestive of protein synthesis during early pregnancy. Presumably, most of the protein synthesis is membranogenesis (Carithers and Green, 1972 b). The mitochondria of early pregnancy were most like the changes described by Carithers (1976) following stimulation with gonadotrophin. Enlarged spherical or oval mitochondria, with tubular cristae, dense matrix and electron-dense granules were common. Cup-shaped mitochondria were intimately associated with lipid vacuoles and cisterns of smooth endoplasmic reticulum. These bizzare mitochondrial configurations and the mitochondrial-smooth endoplasmic reticulum-lipid vacuole complex are believed to be important in steroidogenesis (Christensen and Gillim, 1969). During the last half of pregnancy mitochondria assume conventional rod-like forms with lamellar cristae characteristic of cells in general. Osmiophilic changes in interstitial cell lipid vacuoles can be correlated with patterns described for hydroxysteroid dehydrogenase activity. At estrus lipid vacuoles in adjacent cells withon an interstitial gland were seen to vary in osmiophilia. Some cells had vacuoles that were more lucent than others. Usually all the vacuoles within a given cell were homogenously electron dense or lucent. This observation is in agreement with those reported in the literature (Carithers, 1976). Through Days 4, 6 and 10 the lipid vacuoles appeared to become more electron dense. At Day 14 leaching was apparent, and by Day 18 lipid vacuoles were scarce. Those that were present were electron lucent. The variability in the density of droplets may reflect different droplet contents. Osmium tetroxide used in fixation reacts with double bonds and a pale droplet might contain lipids whose fatty acids are relatively saturated or contain more cholesterol (Christensen and Gillim, 1969). Many other factors also play a role in determining the density of lipid droplets, such as the nature of the buffer used with the fixative, or the extent of lipid extraction during preservation (Bjersing, 1967). However, since these factors were carefully controlled in our study (same buffer, same fixation and dehydration schedules), we feel that differences in lipid density may indeed reflect differences in lipid composition at different times in pregnancy. Morphological changes in the Golgi complex during pregnancy are profound. During early pregnancy, Days 4 and 6, the organelle appears to proliferate, so that Day 6 interstitial cells have multiple Golgi complexes. These organelles are intimately associated with vesicles and tubules of the endoplasmic reticulum. These structural observations support the suggestion of Carithers and Green (1972 a) that the organelle is involved in conjugation of the steroid molecule. Golgi profiles regress in number and complexity during later pregnancy. According to Albertini and Anderson (1975), gap junctions or nexuses have a role in dispersion of small molecules, such as cyclic AM P. Albertini and Anderson
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(1975) believe these messenger molecules may synchronize synthesis and release of steroid. If this hypothesis is true, it would appear that gap junctions may play a role in coordinating steroidogenic activity in the interstitial cells during pregnancy. During early pregnancy, cilia were often noted projecting from interstitial cells. The functional significance of these organelles is unknown; however, it has been suggested that such cilia may serve as a type of sensing device and sample the intercellular milieu (Quattropani, 1977). It is generally held that the corpus luteum is the principal source of ovarian progesterone in the rat. However, as few as two corpora lutea will maintain gestation (Kelsey and Meyer, 1960) and it has been suggested that the non-luteal tissues of the ovary secrete hormones essential for the maintenance of gestation (Kraicer et al., 1971). Hypophysectomy results in regression of interstitial steroidogenic cells (Carithers, 1976) and it has been shown that administration of LH to hypophysectomized rats causes interstitial cells to redifferentiate into cells with characteristics of active steroidogenic cells (Carithers, 1976). These observations suggest that the steroidogenic activity of interstitial cells may be dependent on circulating LH during pregnancy. In the present study, highly stimulated steroidogenic cells were present during the first half of gestation, when LH is known to be high (Morishige et al., 1973). By Day 14, LH is dropping (Morishige et al., 1973) and at Day 18, LH is at its lowest point (Morishige et al., 1973). In the present study, regressive structural changes in the fine structure of the interstitial gland were first noted at Day 14 and was pronounced at Day 18. The temporal correlations between high serum LH values and stimulated interstitial cells, and lowered LH values and regressing interstitial cells, prompt the suggestion that during pregnancy, steroidogenic activity of interstitial gland cells is nodulated, at least partially, by LH. In conclusion, alterations in the fine structure of the rat interstitial gland suggest that this is a dynamic, responsive ovarian component during pregnancy. It is likely that LH or other luteotrophic hormones affect the function of this ovarian component during pregnancy. However, one cannot exclude a role for adrenergic nerves in control of the interstitial gland. We have previously demonstrated an adrenergic innervation to the interstitial gland and shown an increase in ovarian norepinephrine as pregnancy progresses. It is hypothesized that adrenergic nerves alone/or in combination with luteotrophic hormones monitor function of this ovarian component. Experiments are currently in progress to test this hypothesis. Acknowledgements. We thank Dan Whitehead for technical assistance and Ms. Judy Marshburn for typing the manuscript. This study was supported by Grant HD 06899 from the National Institute of Child Health and Human Development, United States Public Health Service.
References Albertini, D.F., Anderson, E.: Structural modifications of lutein cell gap junctions during pregnancy in the rat and the mouse. Anat. Rec. 181, 171-194 (1975) Bjersing, L.: On the ultrastructure of granulosa lutein cells in porcine corpus luteum, with special reference to endoplasmic reticulum and steroid hormone synthesis. Z. Zellforsch. 82, 187-211 (1967) Burden, H.W. : Adrenergic innervation in ovaries of the rat and guinea pig. Amer. J. Anat. 133, 455-462 (1972)
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Burden, H.W., Lawrence, I.E., Jr.: The effects of denervation on the localization of As-3/~hydroxysteroid dehydrogenase activity in the rat ovary during pregnancy. Acta anat. (Basel) 97, 286290 (1977) Carithers, J.R.: Ultrastructure of rat ovarian interstitial cells. III. Response to luteinizing hormone. J. Ultrastruct. Res. 55, 96-104 (1976) Carithers, J.R., Green, J.A.: Ultrastructure of rat ovarian interstitial cells. I. Normal structure and regressive changes following hypophysectomy. J. Ultrastruct. Res. 39, 239-250 (1972 a) Carithers, J.R., Green, J.A.: Ultrastructure of rat ovarian interstitial cells. II. Response to gonadotropin. J. Ultrastruct. Res. 39, 251-261 (1972b) Christensen, A.K., Gillim, S.W.: The correlation of fine structure and function in steroid-secreting cells, with emphasis on those of the gonads. In: The Gonads (K.W. McKerns, ed.), pp. 415-479. New York: Appleton-Century-Crofts 1969 Guraya, S.S.: Function of the human ovary during pregnancy as revealed by the histochemical, biochemical and electron microscope techniques. Acta endocr. (Kbh.) 69, 107-118 (1972) Guraya, S.S.: Interstitial gland tissue of mammalian ovary. Acta endocr. (Kbh.) 72 (Suppl. 171), 5-27 (1973) Karnovsky, M.J.: The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35, 213-236 (1967) Kelsey, R.C., Meyer, R.K.: Amount of luteal tissue required for the maintenance of pregnancy in the rat. Proc. Soc. exp. Biol. (N.Y.) 75, 736-739 (1960) Kraicer, P.F., Kisch, E.S., Nussbaum, M.: Role of non-luteal ovarian tissues in the maintenance of pregnancy. Acta endocr. (Kbh.) 66, 462-470 (1971) Lawrence, I.E., Jr., Burden, H.W.: The autonomic innervation of the interstitial gland of the rat ovary during pregnancy. Amer. J. Anat. 147, 81-94 (1976) Morishige, W.K., Pepe, G.J., Rothchild, I.: Serum luteinizing hormone, prolactin, and progesterone levels during pregnancy in the rat. Endocrinology 92, 1527-1530 (1973) Mossman, H.W., Duke, K.L.: Comparative morphology of the mammalian ovary. Madison: U. Wisc. Press 1973 a Mossman, H.W., Duke, K.L.: Some comparative aspects of the mammalian ovary. In Handbook of Physiology. Sec. 7, Vol. II, Part 1 (R.O. Greep, ed.), pp. 389-401. Baltimore: Williams and Wilkins Co. 1973b Pfliiger, E.F.W. : Uber die Eierst6cke der Sfiugetiere und des Menschen. Leipzig (1863) Quattropani, S.L.: Morphology of the serous cyst of the guinea pig ovary. Anat. Rec. 187, 688 (1977) Reynolds, E.G.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Svensson, K.-G., Owman, Ch., Sj6berg, N.-O., Sporrong, B., Walls, B.: Ultrastructural evidence for adrenergic innervation of the interstitial gland in the guinea pig ovary. Neuroendocrinology 17, 4047 (1975) Unsicker, K.: l]ber den Feinbau der Hiluszwischenzellen im Ovar des Schweins (Sus scrofa L.), Z. Zellforsch. 109, 495-516 (1970) Unsicker, K.: Qualitative and quantitative studies on the innervation of the corpus luteum of rat and pig. Cell Tiss. Res. 152, 513-524 (1974)
Accepted June 8, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Ultrastructure of Rat Ovarian Interstitial Gland Cells during Pregnancy Irvin E. Lawrence, Jr., Hubert W. Burden, and Marilyn L. Capps Department of Anatomy, School of Medicine, East Carolina University, Greenville, North Carolina, USA
Summary. The fine structure of the interstitial gland of the rat ovary was studied at estrus and on Days 4, 6, I0, 14 and 18 of pregnancy. At estrus, ovarian interstitial cells have small nuclei with dense irregular clumps of heterochromatin. Mitochondria are small and rod-shaped and have predominantely lamellar cristae. Numerous osmiophilic lipid droplets are present. At Days 4 and 6, nuclear heterochromatin is reduced, and nucleoli are larger and complex. Mitochondria are enlarged and often bizarre-shaped and have tubular cristae. Golgi and smooth endoplasmic reticulum are more conspicuous. At Day 10, prominent ultrastructural features include nuclei with conspicuous heterochromatin, smaller mitochondria with both lamellar and tubular cristae, numerous ribosomes and lipid droplets with decreased osmiophilia. At Days 14 and 18, nuclei have increased heterochromatin, mitochondria are small and have lamellated cristae and an increase in the size and number of lipid droplets occurs. These observations suggest that steroidogenic activity of interstitial cells is highest during the first half of pregnancy and regresses during the last half. It is suggested that the interstitial gland is an important ovarian source of pregnancy hormone(s) during the first half of gestation and that L H may modulate steroidogenic activity in this ovarian component. Key words: Ovary (Rat) - Pregnancy - Interstitial gland - Ultrastructure.
Introduction The interstitial gland of the mammalian ovary possesses ultrastructural, histochemical and biochemical features of steroid-secreting cells (Guraya, 1973; Mossman and Duke, 1973a, b). Previous electron microscopic studies have demonstrated that this ovarian component responds to endogenous and exogenous gonadotrophins (Carithers and Green, 1972 a, b; Carithers, 1976). An adrenergic Irvin E. Lawrence,Jr., Department of Anatomy, School of Medicine, East Carolina University,Greenville,North Carolina 27834, USA
Send offprint requests to:
144
I.E. Lawrence et al.
i n n e r v a t i o n o f the interstitial g l a n d has been d e m o n s t r a t e d b y several investigators (Unsicker, 1970; Burden, 1972; Unsicker, 1974; Svensson et al., 1975; L a w r e n c e a n d Burden, 1976), a n d it has been suggested t h a t in a d d i t i o n to h y p o p h y s e a l influences, steroidogenic activity o f the gland m a y also be affected by local n e u r a l stimuli (Svensson et al., 1975; L a w r e n c e a n d Burden, 1976). T h e p u r p o s e o f the present s t u d y was to d e m o n s t r a t e at the fine structural level the m o r p h o l o g y o f interstitial g l a n d cells d u r i n g pregnancy. These o b s e r v a t i o n s also f o r m a basis for studies o f the response o f interstitial cells to d e n e r v a t i o n d u r i n g pregnancy, to be p u b l i s h e d later.
Materials and Methods Animals. Female nulliparous Sprague-Dawley rats weighing 165 to 220 g were housed in a room with a 14-h light (0500 to 1900 h) and 10-h dark regimen. Estrous cycles were monitored daily between 0800 h and 1000 h by microscopic evaluation of aqueous vaginal lavages. To establish pregnancy, females in proestrus were housed with a male of proven fertility. The day on which spermatozoa were found in the vaginal lavage was designated as Day 1 of pregnancy. Rats in estrus and on Days 4, 6, 10, 14 and 18 of pregnancy were used in this study. ElectronMicroscopy. For fixation, a thoracotomy was performed and the trocar was placed through the left ventricle and into the ascending aorta. The animal was perfused preliminarily with Earle's balanced salt solution. After a briefwash with the salt solution, the animal was perfused with 3 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). The ovaries were dissected, minced and immersed in fixative to achieve a total fixation time of one and one-half to two hours. After a wash in phosphate buffer the tissue was post-fixed in 1% osmium tetraoxide, stained en bloc with uranyl acetate, (Karnovsky, 1967) dehydrated rapidly, and embedded in Maraglas. Thin sections were cut on a Porter-Blum MT 2-B ultramicrotome and stained with 2 % uranyl acetate and lead citrate (Reynolds, 1963). The tissues were viewed with a Philips EM 201.
Results Estrus. Nuclei in interstitial cells o f estrus rats are small a n d often deeply indented. Dense irregular c l u m p s o f h e t e r o c h r o m a t i n line the inner nuclear m e m b r a n e . A single nucleolus is often eccentrically l o c a t e d a d j a c e n t to h e t e r o c h r o matin. N u c l e o l o n e m a a n d p a r s a m o r p h a are present. M i t o c h o n d r i a are small a n d rod-shaped. Some cristae are tubular, b u t m o s t are l a m e l l a r (Fig. 1). The m i t o c h o n d r i a are frequently l o c a t e d in the cell periphery. T h e G o l g i c o m p l e x is c o m p o s e d o f a parallel stack o f saccules with a few associated vesicles. S m o o t h e n d o p l a s m i c reticulum is n o t conspicuous. A few scattered r i b o s o m e s a n d lysosomes are present. L i p i d d r o p l e t s are n u m e r o u s a n d a p p e a r p o l a r i z e d in the cell, occurring n e a r the c a p i l l a r y surface o r a l o n g the b o r d e r a d j o i n i n g connective tissue. M o s t o f the d r o p l e t s are u n i f o r m l y osmiophilic, a l t h o u g h s o m e d r o p l e t s within a single cell m a y be c o m p l e t e l y leached. Intercellular space is usually filled with a flocculent material. Day 4. Cellular a n d n u c l e a r h y p e r t r o p h y are m o s t a p p a r e n t in this group. Nuclei are enlarged a n d spherical, r o u n d to oval in outline, a n d the h e t e r o c h r o m a t i n is reduced. The nucleoli have a s s u m e d a m o r e central p o s i t i o n a n d are larger a n d m o r e
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Fig. 1. Ovarian interstitial cells from an estrus rat. Mitochondria (M) are small and have lamellar cristae. Lipid droplets (L) of varying sizes are present. Aggregates of nuclear heterochromatin (H) are present. The plasmalemmata of two adjacent cells form a tight junction (arrow). • 17,500
complex. The mitochondria are enlarged, and packed with tubular cristae. Some mitochondria are attenuated, and give the appearance o f division (Fig. 2). Other mitochondria are cup-shaped or annular. Sometimes mitochondria are closely associated with lipid droplets and/or smooth endoplasmic reticulum. Some mitochondria appear to be pinching off cytoplasm (Fig. 4) or "engulfing" other mitochondria (Fig. 5). The Golgi complex is larger and often has dilated cisternae. A few polysomes and scattered ribosomes are present. Distinct profiles of smooth endoplasmic reticulum are difficult to recognize. Filopodia of the steroidogenic cells interdigitate within the intercellular space. Nexuses are seen between some of the abutting cellular processes (Fig. 3).
Day 6. The nuclei are similar to Day 4, however, the heterochromatin is reduced. Nucleoli are less frequently seen. Mitochondria are similar in size and number to Day 4, but the number of cristae is reduced and some cristae are now lamellar. Mitochondria are often contiguous to lipid droplets. Lipid droplets are more uniformly osmiophilic. An extensive smooth endoplasmic reticulum occurs and vesicles o f the endoplasmic reticulum surround some of the lipid droplets (Fig. 5).
Fig. 2. A portion of an ovarian interstitial cell from Day 4 of pregnancy. The nucleus is more vesicular, heterochromatin (H) is reduced and a well organized nucleolus (N) is present. Golgi (G) apparatus is enlarged with stacks of saccules and vesicles. Mitochondria (34) are enlarged, have more tubular cristae, may be closely associated with lipid (L) droplets, and often assume complex shapes, x 16,625 Fig. 3. An extensive gap junction (arrows) between the plasmalemmata of two interstitial ceUs. Day 4 of pregnancy. Mitochondrion (34) with tubular cristae. • 23,750
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Fig. 4. Ovarian interstitial cells from Day 6 of pregnancy. An elongate mitochondrion (h4) appears to be in the process of"pinching off' cytoplasm. Other mitochondria appear annular(AM). At this time, there is a close association between mitochondria, smooth endoplasmic reticulum (arrows) and lipid droplets (L). Filopodia (F) enclose intercellular space. • 16,625
Fig. 5. Cytoplasmic features of ovarian interstitial cells from Day 6 of pregnancy. Note the numerous Golgi (G) profiles, extensive smooth endoplasmic reticulum (arrows) often in close association with lipid (L) droplets. Mitochondria are complex. Some appear to be in the process of "engulfing" other mitochondria (1), while other mitochondria (2) appear to have already been "engulfed". • 16,625 Fig.6. Interstitialcell, Day 6 ofpregnancy.Acilium(C) projects into the intercellular space(IC), x 16,625
Fig. 7. Portions of interstitial cells, Day 10 of pregnancy. Mitochondria (M) are rod-shaped and have a dense matrix. Polysomes (P) are present and lipid droplets (L) have a homogenous, slightly osmiophilic matrix. • 23,750 Fig. 8. Cytoplasmic features of interstitial cells, Day 14 of pregnancy. The contents of the lipid droplets (L) are partially leached. Mitochondria (M) are simple rods with both lamellar and tubular cristae. A tight junction (arrow) is present between adjacent cells. • 23,750
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Fig. 9. Portions of interstitial cells, Day 18 of pregnancy. The nucleus is irregular and heterochromatin (/4) is clumped. Lipid droplets (L) are large and mitochondria (M) are small and have lamellar cristae. • 17,500
Sometimes annular mitochondria may surround the endoplasmic reticulum-lipid droplet complex (Figs. 2, 4, 5). Golgi cisternae and vesicles are often associated with the smooth endoplasmic reticulum-lipid droplet complex. Numerous Golgi profiles occur throughout the cytoplasm (Fig. 5). Cilia often project into the intercellular space (Fig. 6). The basal body of these cilia is always within the vicinity of the Golgi complex.
Day 10. The nuclei are large and mostly spherical. The heterochromatin is conspicuous along the nuclear perimeter. A single nucleolus occurs in most cells. Mitochondria are usually small and rod-shaped and frequently located along one edge of the nucleus. Cristae are both tubular and lamellar, and occasionally dense granules occur in the matrix. The Golgi complex is prominent with lamelliform components and associated vesicles. The smooth endoplasmic reticulum is poorly visualized. Many free ribosomes occur. Lipid droplets are less osmiophilic than Day 6 (Fig. 7). Lipid droplets often occur in one pole of the cell near the capillary and/or connective tissue space. The intercellular space is pronounced. The space appears to be empty, although occasional membranous profiles may be present. Coated vesicles in the steroidogenic cells occur along the intercellular space. Cilia that are sometimes present in the vicinity of the Golgi complex extend into the intercellular space. Day 14. Nuclei of many Day 14 interstitial cells are indented. Some nuclei are large and spherical. The heterochromatin is more dense along the inner nuclear
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membrane. The nucleoli are conspicuous and have a distinct nucleolonema and pars amorpha. Mitochondria are small and have lamellated cristae (Fig. 8). Dense granules seldom occur in the mitochondrial matrix. In some cell, lipid droplets are few and the lipid has an electron-lucent center (Fig. 8). There is little smooth endoplasmic reticulum. Polyribosomes are present. On Day 14 there is less intercellular space. Filopodia are more elongate and interdigitated. Some tight junctions exist.
Day 18. Nuclei are smaller, oval to irregular in shape, indented and have dense heterochromatin. The nucleoli are infrequent. Mitochondria are small and have lamellated cristae (Fig. 9). There appear to be few electron dense granules in the mitochondrial matrix. The mitochondria are more random in distribution, often dispersed between lipid droplets. An increase in the number and size of lipid droplets occurs. The lipid is often partially leached. There are free ribosomes, with scant rough endoplasmic reticulum. The Golgi complex is small.
Discussion
Although the interstitial gland has been recognized in the mammalian ovary for over a century (Pfliiger, 1863) the significance and importance of this structure has been largely overlooked by both anatomists and physiologists. In our laboratory we have directed our attention to studies of the interstitial gland during pregnancy (Lawrence and Burden, 1976; Burden and Lawrence, 1977). The present study has demonstrated changes in the interstitial gland during pregnancy and correlates well with our histochemical observations of A S-3fl-hydroxysteroid dehydrogenase (3 flHSD) activity in the rat ovary during pregnancy. The enzyme activity (with pregnenolone substrate), which is interpreted as an index of the capacity of tissue to secrete progesterone, was most pronounced in the interstitial gland through Day 10 (Burden and Lawrence, 1977). By Day 14, activity in the interstitial gland and corpus luteum was similar and by Day 18, the site of greatest 3fl-HSD activity was the corpus luteum (Burden and Lawrence, 1977). Thus at Day 18, the interstitial gland has less capacity to secrete progesterone as determined by 3 fl-HSD activity and the fine structure of the cells suggests decreased steroidogenic activity. The fine structural changes in interstitial gland cells observed during pregnancy are both quantitative and qualitative. Nuclear and cytoplasmic hypertrophy characterize the first half of pregnancy, whereas during the last half of pregnancy, morphological features suggest a functional regression of interstitial gland cells. Contrary to the conclusion of Guraya (1972), qualitative changes appear in mitochondrial granules, the lipid droplets and the intercellular substance. Quantitative changes were characteristic of filopodia, nexuses, and cilia. Carithers and Green (1972a) observed nuclear and cytoplasmic regression following hypophysectomy. Then in sequal, they carefully monitored the stimulatory effects of gonadotrophin on these structures (Carithers and Green, 1972b). These observations led Carithers to conclude that LH is primarily cytoplasmic in its effect (Carithers, 1976). The earliest obvious change was in hypertrophy of the mitochondria. This was succeeded by other cytoplasmic changes
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leading to membranogenesis that was not completed until after the nuclear changes had been completed (Carithers, 1976). Initiation and development of the nuclear changes were prolonged. Interstitial cells during the first half of pregnancy were highly stimulated. In comparison to estrus, the nucleus and nucleolus were greatly enlarged and vesicular. At Day 4, a spherical nucleus, characteristic of active steroid cells, was present. A larger nucleus appeared in Day 6 interstitial cells. A prominent nucleonema and fibrillar substance constituted the nucleoli at this time. And thinly dispersed, peripherally disposed heterochromatin was present through Days 4 and 6 and extended into Day 10. These nuclear changes are suggestive of protein synthesis during early pregnancy. Presumably, most of the protein synthesis is membranogenesis (Carithers and Green, 1972 b). The mitochondria of early pregnancy were most like the changes described by Carithers (1976) following stimulation with gonadotrophin. Enlarged spherical or oval mitochondria, with tubular cristae, dense matrix and electron-dense granules were common. Cup-shaped mitochondria were intimately associated with lipid vacuoles and cisterns of smooth endoplasmic reticulum. These bizzare mitochondrial configurations and the mitochondrial-smooth endoplasmic reticulum-lipid vacuole complex are believed to be important in steroidogenesis (Christensen and Gillim, 1969). During the last half of pregnancy mitochondria assume conventional rod-like forms with lamellar cristae characteristic of cells in general. Osmiophilic changes in interstitial cell lipid vacuoles can be correlated with patterns described for hydroxysteroid dehydrogenase activity. At estrus lipid vacuoles in adjacent cells withon an interstitial gland were seen to vary in osmiophilia. Some cells had vacuoles that were more lucent than others. Usually all the vacuoles within a given cell were homogenously electron dense or lucent. This observation is in agreement with those reported in the literature (Carithers, 1976). Through Days 4, 6 and 10 the lipid vacuoles appeared to become more electron dense. At Day 14 leaching was apparent, and by Day 18 lipid vacuoles were scarce. Those that were present were electron lucent. The variability in the density of droplets may reflect different droplet contents. Osmium tetroxide used in fixation reacts with double bonds and a pale droplet might contain lipids whose fatty acids are relatively saturated or contain more cholesterol (Christensen and Gillim, 1969). Many other factors also play a role in determining the density of lipid droplets, such as the nature of the buffer used with the fixative, or the extent of lipid extraction during preservation (Bjersing, 1967). However, since these factors were carefully controlled in our study (same buffer, same fixation and dehydration schedules), we feel that differences in lipid density may indeed reflect differences in lipid composition at different times in pregnancy. Morphological changes in the Golgi complex during pregnancy are profound. During early pregnancy, Days 4 and 6, the organelle appears to proliferate, so that Day 6 interstitial cells have multiple Golgi complexes. These organelles are intimately associated with vesicles and tubules of the endoplasmic reticulum. These structural observations support the suggestion of Carithers and Green (1972 a) that the organelle is involved in conjugation of the steroid molecule. Golgi profiles regress in number and complexity during later pregnancy. According to Albertini and Anderson (1975), gap junctions or nexuses have a role in dispersion of small molecules, such as cyclic AM P. Albertini and Anderson
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(1975) believe these messenger molecules may synchronize synthesis and release of steroid. If this hypothesis is true, it would appear that gap junctions may play a role in coordinating steroidogenic activity in the interstitial cells during pregnancy. During early pregnancy, cilia were often noted projecting from interstitial cells. The functional significance of these organelles is unknown; however, it has been suggested that such cilia may serve as a type of sensing device and sample the intercellular milieu (Quattropani, 1977). It is generally held that the corpus luteum is the principal source of ovarian progesterone in the rat. However, as few as two corpora lutea will maintain gestation (Kelsey and Meyer, 1960) and it has been suggested that the non-luteal tissues of the ovary secrete hormones essential for the maintenance of gestation (Kraicer et al., 1971). Hypophysectomy results in regression of interstitial steroidogenic cells (Carithers, 1976) and it has been shown that administration of LH to hypophysectomized rats causes interstitial cells to redifferentiate into cells with characteristics of active steroidogenic cells (Carithers, 1976). These observations suggest that the steroidogenic activity of interstitial cells may be dependent on circulating LH during pregnancy. In the present study, highly stimulated steroidogenic cells were present during the first half of gestation, when LH is known to be high (Morishige et al., 1973). By Day 14, LH is dropping (Morishige et al., 1973) and at Day 18, LH is at its lowest point (Morishige et al., 1973). In the present study, regressive structural changes in the fine structure of the interstitial gland were first noted at Day 14 and was pronounced at Day 18. The temporal correlations between high serum LH values and stimulated interstitial cells, and lowered LH values and regressing interstitial cells, prompt the suggestion that during pregnancy, steroidogenic activity of interstitial gland cells is nodulated, at least partially, by LH. In conclusion, alterations in the fine structure of the rat interstitial gland suggest that this is a dynamic, responsive ovarian component during pregnancy. It is likely that LH or other luteotrophic hormones affect the function of this ovarian component during pregnancy. However, one cannot exclude a role for adrenergic nerves in control of the interstitial gland. We have previously demonstrated an adrenergic innervation to the interstitial gland and shown an increase in ovarian norepinephrine as pregnancy progresses. It is hypothesized that adrenergic nerves alone/or in combination with luteotrophic hormones monitor function of this ovarian component. Experiments are currently in progress to test this hypothesis. Acknowledgements. We thank Dan Whitehead for technical assistance and Ms. Judy Marshburn for typing the manuscript. This study was supported by Grant HD 06899 from the National Institute of Child Health and Human Development, United States Public Health Service.
References Albertini, D.F., Anderson, E.: Structural modifications of lutein cell gap junctions during pregnancy in the rat and the mouse. Anat. Rec. 181, 171-194 (1975) Bjersing, L.: On the ultrastructure of granulosa lutein cells in porcine corpus luteum, with special reference to endoplasmic reticulum and steroid hormone synthesis. Z. Zellforsch. 82, 187-211 (1967) Burden, H.W. : Adrenergic innervation in ovaries of the rat and guinea pig. Amer. J. Anat. 133, 455-462 (1972)
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Burden, H.W., Lawrence, I.E., Jr.: The effects of denervation on the localization of As-3/~hydroxysteroid dehydrogenase activity in the rat ovary during pregnancy. Acta anat. (Basel) 97, 286290 (1977) Carithers, J.R.: Ultrastructure of rat ovarian interstitial cells. III. Response to luteinizing hormone. J. Ultrastruct. Res. 55, 96-104 (1976) Carithers, J.R., Green, J.A.: Ultrastructure of rat ovarian interstitial cells. I. Normal structure and regressive changes following hypophysectomy. J. Ultrastruct. Res. 39, 239-250 (1972 a) Carithers, J.R., Green, J.A.: Ultrastructure of rat ovarian interstitial cells. II. Response to gonadotropin. J. Ultrastruct. Res. 39, 251-261 (1972b) Christensen, A.K., Gillim, S.W.: The correlation of fine structure and function in steroid-secreting cells, with emphasis on those of the gonads. In: The Gonads (K.W. McKerns, ed.), pp. 415-479. New York: Appleton-Century-Crofts 1969 Guraya, S.S.: Function of the human ovary during pregnancy as revealed by the histochemical, biochemical and electron microscope techniques. Acta endocr. (Kbh.) 69, 107-118 (1972) Guraya, S.S.: Interstitial gland tissue of mammalian ovary. Acta endocr. (Kbh.) 72 (Suppl. 171), 5-27 (1973) Karnovsky, M.J.: The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35, 213-236 (1967) Kelsey, R.C., Meyer, R.K.: Amount of luteal tissue required for the maintenance of pregnancy in the rat. Proc. Soc. exp. Biol. (N.Y.) 75, 736-739 (1960) Kraicer, P.F., Kisch, E.S., Nussbaum, M.: Role of non-luteal ovarian tissues in the maintenance of pregnancy. Acta endocr. (Kbh.) 66, 462-470 (1971) Lawrence, I.E., Jr., Burden, H.W.: The autonomic innervation of the interstitial gland of the rat ovary during pregnancy. Amer. J. Anat. 147, 81-94 (1976) Morishige, W.K., Pepe, G.J., Rothchild, I.: Serum luteinizing hormone, prolactin, and progesterone levels during pregnancy in the rat. Endocrinology 92, 1527-1530 (1973) Mossman, H.W., Duke, K.L.: Comparative morphology of the mammalian ovary. Madison: U. Wisc. Press 1973 a Mossman, H.W., Duke, K.L.: Some comparative aspects of the mammalian ovary. In Handbook of Physiology. Sec. 7, Vol. II, Part 1 (R.O. Greep, ed.), pp. 389-401. Baltimore: Williams and Wilkins Co. 1973b Pfliiger, E.F.W. : Uber die Eierst6cke der Sfiugetiere und des Menschen. Leipzig (1863) Quattropani, S.L.: Morphology of the serous cyst of the guinea pig ovary. Anat. Rec. 187, 688 (1977) Reynolds, E.G.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Svensson, K.-G., Owman, Ch., Sj6berg, N.-O., Sporrong, B., Walls, B.: Ultrastructural evidence for adrenergic innervation of the interstitial gland in the guinea pig ovary. Neuroendocrinology 17, 4047 (1975) Unsicker, K.: l]ber den Feinbau der Hiluszwischenzellen im Ovar des Schweins (Sus scrofa L.), Z. Zellforsch. 109, 495-516 (1970) Unsicker, K.: Qualitative and quantitative studies on the innervation of the corpus luteum of rat and pig. Cell Tiss. Res. 152, 513-524 (1974)
Accepted June 8, 1977
