Change of inverted thyroid follicle into a spheroid after embedding in a collagen gel

When inverted thyroid follicles in suspension culture are embedded in a collagen gel, there is extensive reorganization of the follicle. To identify intermediate stages in the reorganization, a suspension of inverted follicles was mixed with a cold solution of collagen (0.1 mg/ml) in culture medium and the resultant was warmed and allowed to gel. Prior to embedding, the epithelial cells bounding the lumens formed a monolayer of attenuated cells with their microvilli-bearing surface in contact with the medium. The first change noted was a shrinkage of the lumen in many follicles by 18 h. The cells became cuboidal to columnar. Some of the cells had long sheet-like processes extending into the lumen in contact with those of other cells. In late stages of the reorganization, 48 h, the cells were arranged in a compact spheroid. The spheroids contained two different kinds of colloid-filled lumens, possibly of different origins, one a spherical microlumen, the other very long and narrow in section. The peripheral cells of the spheroid had a smooth plasma membrane (without microvilli) in contact with collagen. Although most of the cells in a section had a microvilli-bearing surface forming part of the boundary of a lumen, it is not certain that all cells were in contact with a lumen.     

Ultrastructure and some other properties of inverted thyroid follicles in suspension culture

Separated thyroid follicles in suspension culture invert in 5% serum. In some, the inversion is not complete in that a small normal follicle persists completely in the interior of an inverted follicle. In inverted follicles the lumens are distended and electron lucent. The bounding epithelial cells are stretched, have relatively few microvilli on the surface toward the medium but they have bundles of oriented microfilaments usually located near the lumen. The cells are connected together by tight junctions. When inverted follicles are punctured, the lumen shrinks, the cells retract and become cuboidal and microvilli reappear. Microfilaments persist at the luminal surface but no longer in oriented bundles. No appreciable extracellular matrix is present at the basal cell surface in contact with the lumen, but matrix is occasionally observed between cells. Since bundles of microfilaments like stress fibers are observed in the cells in suspension culture, the presence of stress fibers in cells in monolayer culture is probably not dependent on attachment but might be a reflection of the spreading of the attached cells.     

Quantitative autoradiographic study of FSH binding sites in prepubertal ovaries of three strains of rats

Iodinated FSH was injected to 18- and 36-day-old rats of 3 strains (03, 04 and 12) with different sensitivity to FSH (12 less than 03 less than 04) and autoradiography was performed on histological sections of the labelled ovaries. Specific labelling was quantified by microphotometry on histological slides, on granulosa cells of individual follicles with different sizes (greater than 80 micron diameter) and qualities. In small preantral follicles (less than 160 micron diameter) the labelling was low and homogeneous within the granulosa; it increased between 18 and 36 days of age in the 3 strains. At 36 days, ovaries were characterized by the presence of large preantral and antral follicles with a higher labelling in the outer layers of granulosa (near the theca), compared to the inner layers. In definitely atretic follicles, a loss of binding sites was detected in the outer layers. In rats of Strains 03 and 04, the number of binding sites for FSH in the outer layers of granulosa of follicles with a diameter of greater than 160 micron increased with follicular size; no change was detected in follicles of Strain 12 rats. The low number of binding sites for FSH and the lack of terminal maturation which characterize the follicles of strain 12 rats can be related to the poor and delayed follicular development, the low sensitivity to exogenous FSH and the low fertility of the animals of this strain.     

A transient luminal chitinous matrix is required to model epithelial tube diameter in the Drosophila trachea

Epithelial tubes are found in many vital organs and require uniform and correct tube diameters for optimal function. Tube size depends on apical membrane growth and subapical cytoskeletal reorganization, but the cues that coordinate these events to ensure functional tube shape remain elusive. We find that epithelial tubes in the Drosophila trachea require luminal chitin polysaccharides to attain the correct diameter. Tracheal chitin forms a broad transient filament within the tubes during the restricted period of expansion. Loss of chitin causes tubular constrictions and cysts associated with irregular subapical cytoskeletal organization, without affecting epithelial integrity and polarity. Analysis of previously identified tube expansion mutants in genes encoding septate junction proteins further suggests that septate junction components may function in tubulogenesis through their role in luminal matrix assembly. We propose that the transient luminal protein/polysaccharide matrix is sensed by the epithelial cells and coordinates cytoskeletal organization to ensure uniform lumen diameter.     

Membrane retrieval in epithelial cells of isolated thyroid follicles.

Follicles from rat and pig thyroid glands were isolated by digestion with collagenase. The epithelial cells of isolated follicles maintain their structural and functional polarity as shown by incorporation of 3H-leucine and autoradiography. To trace the fate of surface membrane, isolated follicles were opened, stimulated with thyrotropin and incubated for various time intervals with cationized ferritin (CF), uncharged dextran, native ferritin (NF), and latex spheres (0.5 mum in diameter) which were either pre-coated with CF or added together with CF. Uncharged dextran and native ferritin did not bind to the luminal cell membrane, were taken up in small amounts and accumulated in lysosomes; anionic NF was not found in Golgi cisternae in contrast to uncharged dextran which occassionally reached a few Golgi stacks. CF bound rapidly and in clusters to the luminal plasmalemma, preferentially to coated pits, was taken up by endocytosis, accumulated in lysosomes after 5 min and reached the Golgi cisternae after 30 min. Latex spheres were taken up by engulfment through fusion of microvilli and reached the lysosomes. CF particles coating the latex spheres may detach at this station and reach the Golgi cisternae. The findings show that the route of small tracers depends on the charge of the tracer, in agreement with results obtained by Farquhar [8]. Vesicles carrying NF can be traced to lysosomes only, whereas vesicles containing uncharged dextran or - more conspicuously -CF also fuse with Golgi membranes. Large tracers (latex beads) reach only the lysosomes, but CF taken up with them may move to Golgi cisternae.     

Absence of KCNQ1-dependent K+ fluxes in proximal tubular cells of frog kidney

The present study was designed to investigate the functional significance of KCNQ1-mediated K+ secretory fluxes in proximal tubular cells of the frog kidney. To this end, we investigated the effects on rapid depolarization and slow repolarization of the peritubular membrane potential after luminal addition of L-phenylalanine or L-alanine plus/minus KCNQ1 channel blockers. Perfusing the lumen with 10 mmol/L L-phenylalanine plus/minus luminal 293B, a specific blocker of KCNQ1, did not modify the rapid depolarization and the rate of slow repolarization. Perfusing the lumen with 10 mmol/L L-alanine plus/minus luminal HMR-1556, a more potent KCNQ1 channel blocker, did not also alter the rapid depolarization and the rate of slow repolarization. Pretreatment (1 h) of the lumen with HMR-1556 also failed to modify rapid depolarization and rate of slow repolarization upon luminal 10 mmol/L L-alanine. Perfusing the lumen with 1 mmol/L L-alanine plus/minus luminal HMR-1556 did not change the rapid depolarization and the rate of slow repolarization. The pretreatment (1 h) with luminal HMR-1556 did not modify the rapid depolarization and the rate of slow repolarization upon luminal 1 mmol/L L-alanine. The pretreatment (1 h) of the lumen with HMR-1556 did not change transference number for K+ of peritubular cell membrane. Finally, luminal barium blunted the rapid depolarization upon application of luminal 1 mmol/L L-alanine. RT-PCR showed that KCNQ1 mRNA was not expressed in frog kidney. In conclusion, the KCNQ1-dependent K+ secretory fluxes are absent in proximal tubule of frog kidney.     

Fine structural aspects of phagocytotic activity in inverted follicle cells of cultured porcine thyroids

Inversion of thyroid follicles took place when they were isolated by collagenase and trypsin and cultured in suspension in Eagle's medium supplemented with 10% fetal calf serum without TSH. The apical surface facing the culture medium contained numerous microvilli and a central cilium, while the luminal surface became flattened. Phagocytotic activity by pseudopods was promoted after addition of TSH to the culture medium. When the inverted follicles were incubated in culture medium containing TSH (50 mU/ml) and human red blood cells, or TSH and polystyrene latex beads (2.02 micron in diameter) for 1-3 h, numerous red blood cells or latex beads respectively were observed to be taken up by the epithelial follicle cells by scanning electron microscopy, as well as conventional thin-section electron microscopy. These results show that the apical surface (culture medium side) of the epithelial cell of the cultured thyroid follicle whose polarity is reversed phagocytoses red blood cells and latex beads.     

Patterns of follicular growth during the four-day estrous cycle of the rat

Female Sprague-Dawley rats underwent laporatomy during metestrus at 70 to 75 days of age or remained untreated to study the effects of surgical stress on follicular growth. Groups of rats were killed on each day of a 4-day estrous cycle, serial sections of the ovaries were prepared histologically and the number and size of follicles with one or more complete layers of cuboidal granulosa cells were determined. Since no differences due to surgery were found, the data were pooled by day of the estrous cycle (17 or 18 rats/day of cycle) for characterization and comparison of size distribution of follicles on different days of the estrous cycle. Follicles were classified as atretic or healthy and divided into groups by increments of 20 micron of diameter for graphing. Data were analyzed by analysis of variance and least squares means. Significant differences were found in the distribution of both healthy and atretic follicles among days of the estrous cycle. At least 21 follicles/ovary were recruited from less than 260 micron into greater than 260 micron in diameter between proestrus and estrus, and the follicles for ovulation were selected by diestrus. A greater number of growing follicles of 70 to 100 micron in diameter were present at diestrus. From the disappearance of follicles greater than 260 micron between estrus and proestrus, it appears that atresia is a very rapid process.     

Calcium dynamics in the peroxisomal lumen of living cells

We here describe the generation of novel, green fluorescent protein-based Ca(2+) indicators targeted to the peroxisome lumen. We show that (i) the Ca(2+) concentration of peroxisomes in living cells at rest is similar to that of the cytosol; (ii) increases in cytosolic Ca(2+) concentration (elicited by either Ca(2+) mobilization from stores or Ca(2+) influx through plasma membrane Ca(2+) channels) are followed by a slow rise in intraperoxisomal [Ca(2+)]; (iii) Ca(2+) influx into peroxisomes is driven neither by an ATP-dependent pump nor by membrane potential nor by a H(+)(Na(+)) gradient. The peroxisomal membrane appears to play a low pass filter role, preventing the organelle from taking up shortlasting cytosolic Ca(2+) transients but allowing equilibration of the peroxisomal luminal [Ca(2+)] with that of the cytosol during prolonged Ca(2+) increases. Thus, peroxisomes appear to be an additional cytosolic Ca(2+) buffer, but their influx and efflux mechanisms are unlike those of any other cellular organelle.     

Ultrastructure of intermediate stages in polarity reversal of thyroid epithelium in follicles in suspension culture

Separated thyroid follicles can be maintained in suspension culture in Coon's modified F-12 medium in 0.5% calf serum. If the serum concentration is raised to 5%, the follicles undergo inversion in 3-5 d. During the process of inversion, epithelial cells can be observed in intermediate stages of polarity reversal. The earliest ultrastructural changes recognized are surface changes in which tight junctions and microvilli appear at the lateral margins of the cell near the medium. Later, changes in the distribution of intracellular organelles occur. The Golgi apparatus shifts towards the end of the cell facing the medium, and lysosomes shift toward the luminal end of the cell. The right junctions and microvilli at the luminal end of the cell disappear sometime after the cytoplasmic organelles rearrange. The luminal colloid disappears only after the surface changes (loss of tight junctions and microvilli) occur at the luminal end of the cell. There appears to be some regulation of the order in which changes occur during polarity reversal of the thyroid epithelial cell.     

Elimination of the nucleus in preovulatory oocytes of the rainbow trout,Salmo gairdneri Richardson (Teleostei)

A process of elimination of the nucleus in oocytes of the rainbow trout displaying eight different stages is described. The phenomenon was observed only in stage III of sexual maturity. Initially, there is a slight shrinkage of the oocyte accompanied by the loss of yolk granules. This is followed by a cytoplasmatic protrusion into the ovarian lumen, which is covered by the follicular wall. Subsequently, the latter is ruptured and the nucleus migrates toward the opening. Finally, the nucleus leaves the follicle together with a portion of cytoplasm and the occasionally undergoes a breakdown into fragments in the ovarian lumen. The relevance of this mechanism in the process of preovulatory follicular atresia is discussed.     

Calcium is transported into the lumen of pig thyroid follicles by fluid phase basolateral to apical transcytosis

The lumen of thyroid follicles contains a high concentration of thyroglobulin, the thyroid prohormone and a high concentration of calcium (Ca²⁺). As thyroglobulin binds Ca²⁺, intraluminal Ca²⁺ is expected to be in free and protein-bound forms. In the present work, we have investigated the mechanism(s) by which Ca²⁺ could enter the lumen of thyroid follicles. ⁴⁵Ca²⁺ uptake studies were carried out on reconstituted pig thyroid follicles (RTF) and pig thyroid cell monolayers (TCM) in primary culture, representing experimental systems with two compartments (cells + lumina) and one compartment, respectively. ⁴⁵Ca²⁺ accumulation in RTF was rapid during the first hour of incubation and then slowly increased. Analysis of the uptake data with a “two compartments” model gave two kinetic constant values: k_-1 = 1.71 ± 0.28 hr^-1 and k_-2 = 0.20 ± 0.05 hr^-1 (n = 10). The slow uptake process accounted for 20–50% of the total RTF-associated Ca²⁺ after 24 hr. ⁴⁵Ca²⁺ uptake by TCM was rapid and reached a stable level within 1–2 hr. Experimental data fitted with a “single compartment” model and gave a k_-1 value of 1.64 ± 0.15 hr^-1 (n = 10) which was not statistically different from the k_-1 obtained for ⁴⁵Ca²⁺ uptake by RTF. We then compared the kinetics of ⁴⁵Ca²⁺ uptake by RTF with the kinetics of transport of fluid phase markers: [¹⁴C]-sucrose and Lucifer Yellow from the medium to the lumen of RTF. [¹⁴C]-sucrose and Lucifer Yellow uptakes by RTF appeared as slow processes compatible with the entry in a single compartment with k_-1 values of 0.32 ± 0.06 hr^-1 (n = 3) and 0.23 ± 0.015 hr^-1 (n = 3), respectively. These values were not significantly different from the k_-2 value obtained for ⁴⁵Ca²⁺ uptake by RTF. These data suggest that thyroid follicles would possess two independent Ca²⁺ compartments: cells and lumen, and that the entry of Ca²⁺ into the lumen of follicles probably could take place by fluid phase basolateral to apical transcytosis. J. Cell. Physiol. 171:43–51, 1997. © 1997 Wiley-Liss, Inc.     

Expression and activity of gamma-glutamyl transpeptidase in the rat epididymis.

Following Northern analysis, GGT mRNA was found predominantly within the caput epididymides and kidney. The size of mRNAs for kidney, caput, corpus, and ductus deferens were 2.2, 2.3, 2.2, and 2.3 kb, respectively, whereas cauda showed a doublet of 2.2 and 2.3 kb. GGT transpeptidation and hydrolytic activity within epididymal luminal fluids collected by micropuncture showed caput = corpus greater than cauda and corpus greater than caput greater than cauda, respectively. Caput luminal GGT transpeptidation activity was significantly inhibited by serine-borate and was optimal at pH 8.0. The calculated Km and Vmax values for hydrolysis of GSH by caput luminal GGT were 0.06 microM and 2.19 nmoles/min/microliters luminal fluid at pH 8.5 compared to 0.49 microM and 0.49 nmoles/min/microliters luminal fluid, respectively, at the physiological pH 6.5 of caput fluid. These studies would suggest that the epididymis can control the activity of luminal GGT by pH. Lower Km (0.12 microM) and higher Vmax (1.13 nmoles/min/microliters luminal fluid) values were also calculated when GSSG was used compared to GSH. Results from Triton X-114 partitioning experiments suggest that luminal GGT probably exists in both membrane bound and nonmembrane bound forms. Western blot analysis of proteins within epididymal luminal fluids revealed both subunits of GGT in all epididymal regions studied. However, two lower molecular bands, approximately 22 kDa and 21 kDa, were also observed in cauda fluid. It is suggested that as GGT is transported along the epididymal duct it undergoes degradation, which accounts for its loss of activity in the distal epididymal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-cell interactions in the process of differentiation of thyroid epithelial cells into follicles: a study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles

Thyroid cells, cultured in the presence of thyroid stimulating hormone, reorganized within 36-48 hr into follicular structures, the in vitro reconstituted thyroid follicles or RTF. By microinjection of fluorescent probes either into the neoformed intrafollicular lumen (IL) or into cells forming the follicles, we have studied the development and some functional properties of cell-cell contacts involved in a) the formation of the thyroid follicular lumen and b) the communication between thyrocytes within the follicle. The probes were compounds of either low (Lucifer Yellow: LY) or high molecular weight (Dextran labeled with fluorescein: FITC-Dextran and Cascade Blue conjugated to bovine serum albumin: CB-BSA). LY microinjected into IL of 2-9-day-old RTF was seen to label circular spaces with a diameter ranging from 10 to 100 microns. The cells delimiting the IL remained unlabeled. The fluorescent dye remained concentrated in IL for up to 24 hr. FITC-Dextran or CB-BSA microinjected into IL behaved as LY; the probes were restrained into the lumen. A 2 hr incubation of RTF with iodide induced alterations of the structure of IL; an effect mediated by an organic form of actively trapped iodide. A 15-30 min incubation of RTF in a low CA2+ medium caused the opening of IL visualized by the progressive decrease of the fluorescence of probes preinjected into the lumenal space. The same but more rapid effect was obtained by microinjection of EGTA into the IL. The low Ca2(+)-dependent opening of IL was also demonstrated by the release into the medium of thyroglobulin present in IL. Microinjection of LY in a cell involved in the follicle structure led to the rapid labeling of the other cells forming the follicle but LY did not penetrate the IL. Unlike LY, the distribution of FITC-Dextran or CB-BSA injected into cells delimiting the lumen was restricted to the microinjected cells. Alterations of medium or intralumenal Ca2+ concentration which caused the opening of IL did not affect the cell-to-cell transfer of LY. By using fluorescent probe microinjection, we show that the in vitro thyrocyte histiotypic differentiation leads to the reconstitution of functional intercellular junctions: tight junctions insuring the tightness of the neoformed lumen and gap junctions mediating the cell-to-cell exchange of small molecules. The structure of the thyroid follicles appears to be under the control of both extracellular and intralumenal Ca2+ concentrations.     

Effects of guanylin and uroguanylin on rat jejunal fluid and electrolyte transport: comparison with heat-stable enterotoxin

The effects of rat guanylin, human guanylin, human uroguanylin and STa on net fluid and electrolyte transport in the closed jejunal loop were compared in anesthetized rats. STa administered into the lumen caused a concentration-dependent (10(-8) to 10(-6) M) inhibition of net fluid and NaCl absorption in the jejunal loop. Uroguanylin had a similar but weaker effect than STa. Both rat and human guanylin inhibited fluid and NaCl absorption only at 10(-6) M. Their order of potency was STa > human uroguanylin > rat guanylin = human guanylin. Changing the luminal pH from 5 to 8 failed to affect the action of guanylin on fluid absorption. Both STa and uroguanylin, but not guanylin, increased the luminal pH by stimulating bicarbonate secretion. Pretreatment of the jejunal loop with guanylin (10(-6) M) 5 min before the instillation of STa (10(-7) M) significantly reduced the inhibitory effect of STa on fluid absorption. It is concluded that guanylin and uroguanylin administered into the rat jejunal lumen have an STa-like action on fluid and electrolyte transport. Guanylin may act as an endogenous antagonist of STa in the rat jejunum and prevent excessive fluid loss by STa.     

Transcytosis in thyroid follicle cells: Regulation and implications for thyroglobulin transport

In order to analyze quantitatively the translocation of plasma membrane during endocytosis and transcytosis and the regulation of these processes in thyroid follicle cells, the apical cell surfaces of resting and TSH-stimulated inside-out follicles were labeled with cationized ferritin. Morphometric analyses showed that the rates of endocytosis and transcytosis are TSH-dependent. More interestingly, whereas the effect of TSH on endocytosis was transient (with a maximum at 16 min), the effect on transcytosis continued to increase until the end of the experiment (i.e, 70 min). During 1 h of endocytosis, the fraction of membrane involved in transcytosis increased by a factor 4 upon TSH stimulation, corresponding to about 12% of the internalized apical plasma membrane area. Cooling to 15 degrees C slowed down, but did not block endocytosis entirely, whereas transcytosis and transfer to lysosomes were totally inhibited In order to quantitate transcytosis of thyroglobulin (TG) and to ascertain whether this molecule undergoes cleavage during transcytosis, inside-out follicles were incubated in a medium containing 3H-labeled TG in the presence of TSH; upon washing and reopening of follicles, the luminal fluid containing TG after transcytosis was found to contain about 10% of the total radioactivity taken up by follicle cells. Transcytosed TG proved to be unmodified with respect to its electrophoretic mobility. We conclude that (i) the fraction of transcytosed TG corresponds approximately to the fraction of membrane involved in this process, (ii) TG does not undergo cleavage during transcytosis, (iii) endocytosis and transcytosis are regulated by TSH but differ in their kinetics after stimulation, and (iv) transcytosis is affected by temperature in a similar way as transfer to lysosomes, suggesting the existence of a common gating step for both pathways.     

Ovarian structure of the marsupial frog Gastrotheca riobambae (Fowler)

The mature ovary of Gastrotheca riobambae presents large oocytes (3 mm in diameter) of pale yellow color. After ovulation and the beginning of embryonic incubation, the empty postovulatory follicles can be recognized in the ovary for about 30 days. The granulosa of these follicles never fills the follicular lumen and this cavity becomes filled with fluid during the first five days of incubation. Later, at 18 days of incubation, the lumen is mostly empty and contains few cells of the granulosa. Shrinkage results in the disappearance of the follicular cavity by approximately the thirtieth day of incubation. The thecae are thick and become separated by a space. This space is filled progressively with cells, blood capillaries and fluid. After the thirtieth to fortieth day of incubation, these follicles become regressive and disappear. The postovulatory follicles of Gastrotheca may correspond to corpora lutea. The evidence suggests that pouch vascularization and the formation of embryonic chambers of pouch tissue may be under ovarian control. In addition, the process of vitellogenesis is influenced by incubation, as most growth of the ovarian oocyte occurs after birth of the tadpoles. Follicular atresia is common and is similar to that of other frogs.     

Fluctuations in follicular structures of rat thyroid glands during 24 hours: fine structural and morphometric studies

The thyroid follicles of adult male Wistar rats were examined at six evenly spaced times over 24 hr with a morphometric technique. Follicular structures were subjected to distinct variations during 24 hr, with respect to volume and numerical densities of follicles in the thyroid gland, and diameters, volumes, cell numbers, and luminal surface areas of individual follicles. Variations in follicular structures were divided into two phases: a large follicular phase at 1200, 1600, and 2000 hr and a small follicular phase at the other times. Although volume densities of follicles in the gland varied with a small amplitude, diameters, volumes, and cell numbers of individual follicles exhibited distinct fluctuations during 24 hr. Numerical densities of follicles in the gland changed distinctly during the small follicular phase as well. Degenerating follicular cells appeared in the follicular lumen especially at 1600 hr. No mitotic follicular cells were found throughout the experiment. Furthermore, one to three follicular cells of two adjacent follicles were often in contact with each other at 0400, 0800, and 1200 hr, and these follicles were lined by the common basement membranes. These results suggest that the variations in follicular structures during the small follicular phase occur in the form of follicle separation and fusion. Moreover, the morphological and morphometric variations in follicles reflect those in subcellular structures of follicular cells previously reported by us.     

Morphometric analysis of follicular dynamics in pregnant and pseudopregnant rats

Morphometric analysis of the follicle population greater than or equal to 100 X 10(5) micron 3 or a mean diameter of greater than or equal to 275 micron and assessment of the rate of atresia in ovaries of pregnant and pseudopregnant rats revealed no evidence for the presence of rhythmic follicular maturation during the prolonged dioestrous period. During the first 4-5 days of the dioestrous period, follicles developed to preovulatory size (volume class 5, i.e. greater than or equal to 1000 X 10(5) micron 3 = diam. greater than or equal to 576 micron) reaching the normal number of ovulating follicles in cyclic animals in pregnant rats, but only half that number in pseudopregnant rats. These follicles collapsed on the 5th to 8th days of the dioestrous period and full numbers of preovulatory follicles were not found thereafter until the end of pregnancy and pseudopregnancy. Follicles of smaller sizes (classes 1-4: 100-999 X 10(5) micron 3), however, were present throughout the prolonged dioestrous period. The rate of atresia in the follicle population had increased by the 2nd day and remained from then on at 26.5 +/- 4.5% in the pregnant and 34.3 +/- 1.9% in the pseudopregnant rats. Atretic follicles in the advanced stages of atresia, mostly derived from follicles of classes 1-3, persisted and accumulated at the end of the dioestrous period. The continuous presence of follicles and the constant rate of atresia during the dioestrous period indicate continuous follicular replacements and refute the idea of follicular quiescence during pregnancy and pseudopregnancy. Copulation and electrical stimulation of the cervix seemed to reduce the formation of the new crop of follicles the next morning and the pool of small antral follicles normally maintained after oestrus in cyclic animals. Nevertheless, the smaller crop and pool of follicles seemed able to provide a sufficient number of preovulatory follicles at the end of pregnancy and a sufficient number of ovulations at the end of pseudopregnancy.     

Cysteine proteinases mediate extracellular prohormone processing in the thyroid

Thyroglobulin, the precursor of thyroid hormones, is extracellularly stored in a highly condensed and covalently cross-linked form. Solublization of thyroglobulin is facilitated by cysteine proteinases like cathepsins B and K which are proteolytically active at the surface of thyroid epithelial cells. The cysteine proteinases mediate the processing of thyroglobulin by limited extracellular proteolysis at the apical plasma membrane, thereby rapidly liberating thyroxine. The trafficking of cysteine proteinases in thyroid epithelial cells includes their targeting to lysosomes where they become maturated before being transported to the apical plasma membrane and, thus, into the extracellular follicle lumen. We propose that thyroid stimulating hormone regulates extracellular proteolysis of thyroglobulin in that it enhances the rate of exocytosis of lysosomal proteins at the apical plasma membrane. Later, thyroid stimulating hormone upregulates thyroglobulin synthesis and its secretion into the follicle lumen for subsequent compaction by covalent cross-linking. Hence, cycles of thyroglobulin proteolysis and thyroglobulin deposition might result in the regulation of the size of the luminal content of thyroid follicles. We conclude that the biological significance of extracellularly acting cysteine proteinases of the thyroid is the rapid utilization of thyroglobulin for the maintenance of constant thyroid hormone levels in vertebrate organisms.