Effect of bovine pituitary hormone preparations on newt lens regeneration in vitro: Stimulation by thyrotropin

DEVELOPMENTAL

BIOLOGY

81,23-35

(1981)

Effect of Bovine Pituitary Hormone Preparations on Newt Lens Regeneration in V&o: Stimulation by Thyrotropin ROBERTCUNY Department

of Zoology, Received April

University

AND SARA

of Alberta.

E. ZALIK

Edmonton.

Alberta,

14, 1980; accepted in revised form

TSG 2E9, Callada

Ju.ne 9, 1980

The anterior lobe of the pituitary gland can stimulate lens regeneration from the dorsal iris in the newt Notophthalmus viridescens. We have studied the effect of pituitary hormone preparations on this process. Dorsal irises were cultured for 20 days in diluted Medium 199 supplemented with 10% fetal calf serum. Bovine thyrotropin TSHB8 at concentrations of 30 to 3000 fig/ml significantly stimulated lens regeneration in these dorsal irises. Well-developed lenses, up to stage 9, were formed, in which y-crystallin, a protein specific for lens fibers of young lenses, was detected by immunofluorescence. Additionally, the mitotic index was 5.5 times elevated in these explants when compared to their controls. Lutropin LH-BlO at concentrations of 30 to 3000 Fg/ml, prolactin PRL-B4 at concentrations of 23 to 1600 pg/ml, and porcine adrenocorticotropin ACTH-6002 at concentrations of 3 to 300 rg/ml did not stimulate lens regeneration. A weak stimulation of lens formation was observed in iris cultures with 2700 fig/ml of follitropin FSHBl or 3000 pg/ml somatotropin GH-B18, but not at concentrations of 30 pg/ml. Our results suggest that the inherent ability of the dorsal iris to form lens can be activated by the bovine thyrotropin preparation TSH-B8. INTRODUCTION

the lens. It has been suggested that the neural retina plays an important stimulatory role in this process (Spemann, 1905). If the neural retina is removed after lentectomy, lens regeneration is delayed until the neural retina starts regenerating (Wachs, 1920; Zalokar, 1944a; Stone and Steinitz, 1953; Stone, 1957; Hasegawa, 1965; Reyer, 1971). Lens regeneration only occurs in iridocorneal complexes grafted on to the head of the newt if the neural retina has regenerated from the ciliary epithelium (Zalokar, 1944a). More direct evidence for the importance of the neural retina has emerged from experiments by Connelly et al. (1973) and Yamada et a.1.(1973). These investigators cultured dorsal irises in close apposition to larval frog retinae, and lenses of different developmental stages developed. The ability to stimulate lens regeneration is not confined to the neural retina. Extraocular tissues, such as spinal nerves of regenerating limb blastemas (Reyer et al., 1973) and in particular the pituitary gland (Zalokar, 1944b), have been reported to promote lens regeneration. Zalokar first reported that cultured lentectomized eyes, in which the neural retina tended to degenerate, only regenerated their lenses if a pituitary gland was inserted into their posterior chambers, and Powell and Segil (1976) observed the regeneration of secondary lenses in nonlentectomized eyes in vivo if pituitary glands were inserted in the anterior chambers. Lenses also regenerated from irises cultured in vitro in apposition to newt or frog pituitary glands (Connelly et al., 1973).

Species of the family Salamandridae (Urodela) have the remarkable ability to regenerate large portions of their eyes in the adult state (Bonnet, 1781). Particular attention has been paid to the regeneration of an entirely new second lens, which regularly develops from the dorsal pupillary margin of the iris after the original lens has been removed (Wolff, 1895). It has become evident (Colucci, 1891; Yamada et al., 1973) that the pigmented dorsal iris epithelial cells of neuroectodermal origin are the source of the secondary lens. This contrasts with the embryonic situation whereby head ectodermal cells are the source of the primary lens cells (Rabl, 1898). The phenomenon of lens regeneration has been reviewed recently (Reyer, 1977; Yamada, 1977). Iris epithelial cells, cultured on plastic in serum-containing media, have the capacity to form dense clusters of lens protein-containing cells or lentoid bodies (Horstman and Zalik, 1974; Eguchi et al., 1974; Yamada, 1977). The conversion of cell type that occurs under these conditions differs in many ways from lens regeneration in vivo. A slightly longer time is required for lentoid body formation in cell culture than for lens regeneration in the eye and no morphological organization into lens epithelial and lens fiber regions is evident. Furthermore, ventral iris cells, which cannot normally form a lens in vivo, will readily form lentoid bodies in cell culture. In the eye, dorsal iris epithelial cells need to be activated to form a lens, for example by the removal of 23

0012-1606/81/010023-13$02.00/O Copyright All rights

0 1981 by Academic Press. Inc. of reproduction in any form reserved.

24

DEVELOPMENTALBIOLOGY

VOLUME81, 1981

TSH-BE irises;';g;;;i

18

j

3,

, r;

4,

CONTROLS 5

6,

1

7,

8;

.

ir,se?$4y5m667mBi

13000

ug/ml*

15

FIG. 1. Lens regeneration stages of dorsal irises after 20 days of culture with or without bovine thyrotropin preparation TSH-BS; 3000 pgg/ml TSH-BS = 10.5 IU. The line between lens regeneration stages 2 and 3 represents the border between specific and nonspecific lens regeneration stages as defined under Materials and Methods. The numbers above the histograms represent the number of individual irises at that particular regeneration stage. Twenty irises have been cultured, but only the ones harvested are included in the statistical analysis. The frequency of specific lens regeneration stages (stages 3 to 8) is significantly higher in TSH-B&treated cultures than in controls. The significance levels (x2) are: *P < 0.05, ***P < 0.005, "P < 0.10 (almost significant).

In view of the above-mentioned experiments it was of interest to us to determine whether any of the available pituitary hormone preparations would have a stimulatory effect on lens regeneration in cultured newt irises. In this paper we will report that a preparation of bovine thyrotropin can induce lens regeneration in vitro. MATERIALS

AND METHODS

Preparation. of explants. Adult newts (Lee’s Newt Farm) were washed in 0.001% potassium permanganate, anesthetized in 0.1% ethyl-m-aminobenzoate methanesulfonate (Sandoz), and their heads were quickly washed in 70% ethanol and sterile distilled water. All further steps were performed under sterile conditions. The lens was gently pressed out of the eye through a horizontal incision in the cornea. The dorsal half of the cornea was then cut out together with the adhering iris, and transferred to modified amphibian Ringer’s solution, containing 6500 mg NaCl, 140 mg KCl, 120 mg CaClz, 100 mg MgSO,, 1800 mg D-glucose, and 200 mg gentamicin sulfate (Schering or Sigma) per liter of 0.005 M phosphate buffer at pH 7.6. The iris tissue was separated from the cornea1 tissue using scalpel blades, and was transferred into culture medium. In some cases the pituitary gland was also excised. Culture conditions. In each experiment, 20 explants per treatment group were cultured for 20 days in 0.3 ml of medium in plastic organ culture dishes (Falcon), and were gently swirled once a day to prevent attachment. The culture medium was changed daily and con-

sisted of 50% Medium 199 with Earle’s salts (Grand Island), 50 pg/ml L-ascorbic acid (Sigma), 50 pg/ml gentamicin sulfate, 10% dialyzed fetal calf serum (Grand Island), and 40% distilled water; it had a pH of 7.6. The fetal calf serum was dialyzed (molecular weight cutoff of dialysis bags of 12,000to 14,000daltons) for 24 hr against two changes of 40 times its volume of distilled water and its salt concentration was readjusted with modified amphibian Ringer’s salts. Media, sera, and Ringer’s solutions were sterilized by filtration. Cultures were maintained at 23°C in a humidified atmosphere containing about 1% COz. Bovine thyrotropin (NIH-TSH-B8), follitropin (NIHFSH-Bl), and lutropin (NIH-LH-BlO), kindly supplied by U. S. National Institute of Arthritis, Metabolic and Digestive Diseases, were directly dissolved in complete culture medium at pH 7.6. Bovine prolactin (NIH-PRLB4), and somatotropin (NIH-GH-B18) were first dissolved in distilled water at pH 9.5, and the pH was then adjusted to 7.6. Both hormone solutions were combined with undiluted culture medium at a ratio of 2:3 (v/v) to the desired concentrations. Porcine adrenocorticotropin (Sigma A-6002) was directly dissolved in complete culture medium at pH 7.0, and the pH was then adjusted to 7.6. After sterile filtration, aliquots of hormone-supplemented media were stored frozen in liquid nitrogen. Preparation of a.ntibodies. y-Crystallin was purified from leopard frog (Rana pipiens) lenses as described by McDevitt et al. (1969), and Niithiger et al. (1971). Rabbits were injected subcutaneously in the back as follows: 5 mg y-crystallin in 0.1 ml saline were emul-

CUNY AND ZALIK

Pitztitary Hmones

in Lens Regeneration

25

a FIG. 2. Lens regenerates which developed on dorsal irises after 20 days of culture with TSH-B8. (a) Stage 1 of lens regeneration in a control culture. x500. (b) Stage 7 obtained with 300 Kg/ml of TSH-B8. Notice the lens epithelium-like region at the inside of the fiber core (arrow). Stained with hematoxylin-eosin. X506.

sified with 0.9 ml of complete Freund’s adjuvant (Difco). Three weeks later, the same amount was given in incomplete adjuvant. Six and 9 weeks after the first injection, 10 mg of antigen were given in incomplete adjuvant. The antiserum was collected 3 weeks after the last injection and was stored frozen in aliquots. The specificity of the y-crystallin antiserum was tested by immunoelectrophoresis. Thirty micrograms of soluble frog lens protein were subjected to electrophoresis for 3 hr at 5°C in an agar gel (0.16 X 5 X 12 cm) using an 0.005 M barbital-acetate buffer, pH 8.5, and a continuous power supply of 8.3 V/cm and 15 mA. Antiserum was added to a central trough and precipitation arches formed within 2 days at 5°C. Gels were washed in two changes of 0.1 M phosphate-buffered

saline, pH 7.2, for 2 days in the cold, and subsequently in distilled water for 2 hr. They were then dried and stained with Coomassie brilliant blue R (Sigma). Immunojhorescent staininy. Cultured tissues were fixed in Bouin’s fixative for 2.5 hr and washed for 1 hr in several changes of 0.1 M phosphate buffer, pH 7.4, at room temperature. They were then dehydrated in graded ethanol and xylene in the cold, embedded in paraplast at 56”C, and sectioned at 7 pm. Sections were deparaffinized in the cold, and were then hydrated and washed in 0.1 M phosphate buffer, pH 7.4, at room temperature for 10 min followed by two half-hour changes of 0.1 M phosphate-buffered saline, pH 7.4. All further steps were performed at room temperature. Tissue sections were coated with rabbit antifrog y-crystallin an-

26

DEVELOPMENTALBIOLOGY

VOLu~~81.1981

fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin, diluted 1:20 (Microbiological Associates). Two hours later, the sections were washed again in three changes of phosphate-buffered saline for 1 hr, were temporarily mounted in glycerol, and stored in the cold. After microscopic examination of the fluorescent staining patterns, sections were stained with hematoxylin-eosin and mounted in DPX. Analysis of results. Lens regeneration stages were estimated according to Sato (1940), as adapted for Notophth.almus viridescens by Yamada (1967). Stages 1 and 2 are reversible cell activation stages undergone by dorsal as well as ventral iris and do not necessarily indicate the onset of lens regeneration. For the purpose of statistical evaluation we only regarded stages 3 and higher as indicative of lens regeneration. The 2 x 2 table x2 test for independence was applied to compare the frequency of lens regeneration in controls with each experimental treatment group. Irises that had reached stages 3 to 9 were regarded as regeneration positive, while irises which had only attained stages 1 or 2 were regarded as regeneration negative. Some of the 20 irises per treatment group were lost during the experimental handling and were not available for analysis. The relative abundance of y-crystallin in lens regenerates was roughly classified in three groups: (+) only a few weakly fluorescent cells, (++j a cluster of 11 to 50 strongly fluorescent cells, and (+++) a large lens fiber core with over 100 strongly fluorescent cells. FIG. 3. Lens regenerate which developed on a dorsal iris after 20 days of culture in the presence of 3000 rg/ml of TSH-B8. Notice the prevalence of the lens epithelium-like region. X464.

Effect of Bovine Thyrotropin

tiserum, diluted 1:20, which had previously been absorbed with 1% tissue powder suspension of lentectomized frogs at a ratio of 1:l (v/v). After 2 hr of incubation, sections were washed in three changes of phosphate-buffered saline for 1 hr, and coated with

Morphology. Dorsal irises were cultured in the presence of bovine thyrotropin preparation TSH-B8 at concentrations ranging from 3000 to 30 pg/ml (Fig. 1). Lens regenerates, stages 3 and higher, were observed in 27% (5 of 18) of the irises in cultures with 3000 pg/ml TSHB8, and 69% (11 of 16) were regeneration positive in

RESULTS

Preparation

TSH-B8

CONTROLS

TSH-B8

stages 12

3

4

5

6

7

8

9

I

FIG. 4. y-Crystallin accumulation in lens regenerates of dorsal irises after 20 days of culture with or without bovine thyrotropin preparation TSH-B8. (+) Faint fluorescence label on only 1 to 10 cells per lens regenerate, (++) strong fluorescence label in 11 to 50 cells, (+++) strong fluorescence label in over 100 cells. y-Crystallin accumulation is correlated with higher lens regeneration stages, although y-crystallin is not always confined to lenses under culture conditions. The significance level (x2) for the difference in frequency of specific lens regeneration stages (stages 3 to 9) of TSH-B8-treated cultures as compared to controls is: **P < 0.01. See Fig. 1 for further explanation of the histograms.

CUNVANDZALIK

Pituitary

a

i#i A

crystallins

b FIG. 5. Specificity of anti-frog y-crystallin antiserum for frog ycrgstallin. (a) Immunoelectrophoresis of frog aqueous lens extract against anti-frog y-crystallin antiserum only yields one (y) crystallin precipitation arch, whereas (b) anti-frog whole crystallin antiserum yields 3 (nJ.-y) crgstallin precipitation arches.

cultures supplemented with 300 pg/ml of this hormone preparation, as compared to none and 13% (2 of 15) in their respective controls. The lowest concentration of TSH-B8 tested during this study, 30 pg/ml, also stimulated lens regeneration. At this concentration, in two replicate experiments 43% (6 of 14, Fig 1) and 65% (11 of 17, Fig. 4) of the irises developed lens regenerates, compared to 13% (2 of 15, Fig. 1) and 17% (3 of 17, Fig. 4j in their controls. In addition to the significantly higher frequency of lens regenerates from dorsal irises cultured in the presence of TSH-B8, lens differentiation was also further advanced. At the highest concentration of TSH-B8, 3000 pg/ml, lens regeneration stages characterized by the beginning of lens fiber development (stage 6, Fig. 3) were reached, while only the iris depigmentation stage 2 was attained in controls. Similarly, at a TSH-B8 concentration of 300 pg/ml, regenerates up to stage 8, characterized by the beginning of secondary lens fiber cell development, were formed. In contrast, control irises reached stage 3 only, characterized by a thickening and depigmentation of cells at the dorsal iris margin. With the lowest TSH-B8 concentration tested, 30 pg/ml, a lens regenerate, similar to the one illustrated in Fig. 2b, with a primary fiber hillock characteristic of stage 7, was obtained in the first experiment, and a lens regenerate of stage 9 with a large fiber core with secondary fiber cells was obtained in the replicate experiment (Fig. 7). These results contrast with those of the controls, in which only regenerates of stage 3 were obtained in the first experiment and one lens vesicle of stage 4 in the second experiment.

27

Hormones i,n Lens Regeneration

No mitotic activity is detectable in the undisturbed dorsal iris (Yamada and Roesel, 1971). It reaches maximal values between 7 and 25 days after lentectomy (Eisenberg and Yamada, 1966j, from the period of depigmentation of dorsal iris cells up to the time of the detachment of the young lens from the iris margin. We determined whether the average mitotic activity in irises stimulated with TSH-B8 would similarly be increased, by estimating the mitotic index of the irises after 20 days of culture (Fig. 9, same irises as in Fig. 1). Although there was large variability in TSH-B8treated irises, the average mitotic index was 5.5 times higher than that of controls. This mainly reflects the index of the iris cell population not involved in the formation of the lens, since these cells constitute a relatively large proportion of the explant. The distribution of the mitotic activity in the lens regenerates is similar situation, with the lens to that found in the in do fibers having the lowest mitotic activity and the remaining lens cells of the regenerate attaining the highest mitotic index. Presence ofy-crystallin. y-Crystallin is the last lensspecific protein to appear during lens regeneration in the newt (Takata et al., 1966), and in young lenses in sdtu it is confined to the lens fiber cells. We have tested for the presence of y-crystallin in lens regenerates as an additional criterion for lens differentiation, by using an immunofluorescence technique. That antibodies directed against frog y-crystallin also react specifically with newt y-crystallins has been documented (Nbthiger et ad., 1971). We have tested the monospecificity of our anti y-crystallin antibodies by immunoelectrophoresis (Fig. 5). Although three different crystallins were present in frog aqueous lens extracts, the antibody formed only one precipitation arch corresponding to y-crystallin. In addition, when sections of newt eyes with 30-day lens regenerates of stage 11 were stained, only the lens fiber core was fluorescent (Fig. 6), but not the lens epithelium or any other part of the eye. The presence of y-crystallin was found to be well correlated with the morphologically estimated lens regeneration stages (Figs. 4,7,8) although a few deviations were observed. In V~VO,regenerates up to the lens vesicle stage 4 do not contain y-crystallin. Under the present culture conditions, however, groups of cells positive for y-crystallin can occur in regenerates classified as stage 2. Effect of Bovine Follitropin

Preparation

FSH-Bl

Morphology. Bovine follitropin FSH-Bl was used at concentrations between 2700 and 30 pg/ml. Only the highest concentration significantly induced lens regeneration, in 29% (4 of 14) of the irises, while no lens regeneration was evident in the controls (Fig. 10). The

28

DEVELOPMENTALBIOLOGY

VOLUME81, 1981

FIG. 6. Specificity of antifrog y-crystallin antiserum for young newt lens fiber cells in v&o. Eye cross sections with stage 11 lenses, 30 days after lentectomy, were stained with anti-frog y-crystallin antiserum or unimmunized serum. (a) Transmission light micrograph, and (b) corresponding immunotluorescent light micrograph with unimmunized serum. No fluorescence is detectable. (c) Transmission light micrograph, and (d) corresponding immunofluorescent light micrograph with anti-frog y-crystallin antiserum. Note that the lens epithelium remains unstained (arrow). a-d X27.3.

frequency of 22% (4 of 18) lens regenerates at 300 pg/ ml and 10% (2 of 20) at 30 pg/ml was not significantly different from that of 13% (2 of 15) in both of their controls. Only early stages of lens regeneration were obtained with FSH-Bl, the most advanced being a lens vesicle of stage 4.

Effect of Bovine Lutropin Preparation

LH-BlO

Morphology. Bovine lutropin preparation LH-BlO was used at concentrations of 3000 to 30 pg/ml. No stimulatory effect on lens regeneration could be observed (Fig. 11). Although 13% (2 of 15) of the control irises had thickened dorsal iris margins of stage 3, none of

the irises cultured in the presence of 3000 pg/ml of LHBlO started lens regeneration. With a concentration of 270 &g/ml the frequency of lens regeneration was only 6% (1 of 15) as compared to no regeneration in the controls, and with LH-BlO at 30 gg/ml, 17% (2 of 12) of the irises compared to 13% (2 of 15) in controls underwent early regeneration. Only the very early lens regeneration stage 3 was found in LH-BlO-supplemented cultures.

Effect of Bovine Somatotropin Preparation Morphology. Three concentrations tropin

preparation

GH-B18

GH-B18

of bovine somatowere used, ranging from

CUNY AND ZALIK

Pituitary

Hormones in Lens Regeneration

29

FIG. 7. &rystallin accumulation in a lens regenerate of stage 9 which developed on a dorsal iris after 20 days of culture in the presence of 30 pg/ml of TSH-BS. (a) Stained transmission light micrograph, and (b) corresponding immunofluorescent light micrograph stained with anti-frog y-crystallin antiserum. (a, b) X253. Note that the lens epithelium-like cells are not stained (arrow).

3000 to 30 pg/ml. Although some relatively advanced stages, up to a lens vesicle of stage 5, were obtained at the highest concentration, they were not very frequent and therefore not statistically significant (Fig. 12). With 3000 pg/ml, 18% (4 of 17) lens regeneration was stimulated as compared to 13% (2 of 15) in controls. With 300 pg/ml, lenses started to regenerate in 7% (1 of 15) of the irises as compared to none in controls, and with 30 pg/ml of GH-B18 6% (1 of 18) lens regeneration occurred as compared to 13% (2 of 15) in the controls.

Effect of Bovine P,rolactin

Prepa.ration PRL-B4

Morph.ology. Bovine prolactin preparation PRL-B4 did not significantly affect lens formation at concen-

trations of 1600 to 23 pg/ml (Fig. 13). No lens regenerates were present in any of these cultures, although some thickened dorsal iris margins of stage 3 occurred in the controls.

Effect of Porcine Ackenocorticotropin ACTH-6002 Morphology. At concentrations between 300 and 3 pg/ml of this hormone preparation, no significant level of lens regeneration was observed (Fig. 14). No lens regeneration was induced at all at 300 and 3 pg/ml, and at 30 pg/ml only 20% (3 of 15) of the irises had lens regenerates, the most advanced being an early lens vesicle at stage 4. In the respective control cultures, lens regenerates developed up to stage 3 in 13% (2 of 15) of the irises.

DEVELOPMENTAL BIOLOGY

VOLUME 81, 1981

FIG. 8. y-Crystallin accumulation in a lens regenerate of stage 6 which developed on a dorsal iris after 20 days of culture in the pressonce of 30 pg/ml of TSH-BS. (a) Stained transmission light micrograph, and (b) corresponding immunofluorescent micrograph stained with antifrog y-crystallin antiserum. Note that the lens epithelium-like cells are not stained and tend to repigment (arrow). (a, b) X240.

Effect of Newt Pituitary Gland Morphology. Lens regeneration

irises

15 CONTROLS

14 TSH-BB 30 pgfml

FIG. 9. Mitotic activity in dorsal irises after 20 days of culture with or without 30 pg/ml TSH-B8. The average estimated mitotic index (mitotic figures/total number of nuclei) is significantly (Wilcoxon’s rank test) elevated in TSH-B8supplemented cultures when compared to the controls; P 6 0.01. The bars above the histograms represent one standard deviation. In irises which had lens regenerates, mitotic indices could be estimated separately for lens fiber cells, lens epithelium-like cells, and dorsal iris cells. The mitotic activity pattern, with the highest activity in lens epithelium-like cells and the lowest activity in lens fiber cells, corresponds well to the activity patterns described for lens regenerates in wiwo.

has previously been obtained when pituitary glands were cultured with iridocorneai complexes (Connelly et al., 1973). In order to test whether dorsal iris sectors responded to the newt pituitary gland under the present culture conditions dorsal irises were cultured with this tissue. Under our conditions, 44% (7 of 16) and 46% (6 of 13) lens regeneration occurred in two replicate experiments, as compared to none or 13% (2 of 15) in their controls (Fig. 15). The most advanced lens regeneration stages were lens vesicles at stage 4, or at beginning lens fiber development (stage 6), whereas in the controls only depigmentation stage 2 or the iris margin thickening of stage 3 were achieved. Thus, under these conditions, the irises were able to respond to the influence of the pituitary gland with a significantly augmented frequency of lens regeneration. DISCUSSION

In this report we have demonstrated ration of bovine thyrotropin, TSH-B8,

that a prepacan stimulate

Pituitary

CUNYANDZALIK

31

Hormones in Lens Regenera.tion CONTROLS

FSH-El

13

18

300

ugfrnl

10

15

15 20

30 ughl

15

10

FIG. 10. Lens regeneration stages achieved by dorsal irises after 20 days of culture with or without bovine follitropin preparation FSH-Bl; 2700 Kg/ml FSH-Bl = 1.3 NIH-U/ml. Except for cultures supplemented with the highest concentration of FSH-Bl, no significant differences t& have been found. *P < 0.05; no asterisk, not significant. See Fig. 1 for further explanation of the histograms.

lens regeneration from irises cultured in vitro. Several hormone preparations are known to stimulate growth and differentiation of lens cells. Bovine thyrotropin, ovine lutropin, tri- and tetraiodothyronine (Rothstein and Worgul, 1973), and bovine somatotropin (Rothstein et al., 1976), when injected intraperitoneally, stimulate the mitotic activity in lens epithelia of the frog. Crude adrenocorticotropin preparations (Takano, 1962) accelerate lens regeneration from the dorsal iris in the newt when injected intraperitoneally. Insulin stimulates lens fiber differentiation in cultured embryonic chick lens epithelia (Piatigorsky, 1973; Milstone and Piatigorsky, 1977), and mitosis in adult rabbit lens epithelia (Reddan and Wilson, 1978). Dexamethasone also stimulates lens fiber cell elongation in cultured calf lens epithelial cells (Arruti and Courtois,

1978). However, insulin does not appear to stimulate cell-type conversion in the iris of the newt (Takano, 1962; Connelly, 1977c), although it can substantially elevate the mitotic rate in cultured newt dorsal irises (unpublished results). Beebe et al. (1980) have isolated a protein, lentropin, from the vitreous body of the chick embryo which promotes lens fiber differentiation in lens epithelia. It is important to point out that the above-mentioned compounds stimulate further differentiation of already committed cells and attempts to induce cell-type conversion in. viva in the iris with chemical preparation, such as prostaglandins (Connelly, 1977a), in vitro with cortisole, tetraiodothyronine, and prolactin (Connelly, 1977c), and in viva with mammalian sex hormones and hydrocortisole (Takano, 1962) have been unsuccessful to date.

LH-B10

CONTROLS

stages 12 frfse

stages 3

4

5

6

7

12

8

,4

19

r;l

12

/

4

5

irises ,

+

3

1

13000

ug/ml

15

w

.

1

30 fig/ml

15

w

FIG. 11. Lens regeneration stages achieved by dorsal irises after 20 days of culture with or without 3000 pg/ml LH-BlO = 2.2 NIH-U. Differences were not significant. See Fig. 1 for further explanation

.

6

7

8 1 j

1

bovine lutropin preparation of the histograms.

LH-B10;

32

DEVELOPMENTAL BIOLOGY

VOLUME 81, 1981 CONTROLS

GH-618 ,Yges* irises

1

3,

4

5

6

stages

a

7

12

3

4

5

6

7

a

iriSeS\

12

17

3000

ug/ml

300

uglml

12 15

FIG. 12. Lens regeneration stages achieved by dorsal irises after 20 days of culture with or without bovine somatotropin preparation GHB18; 3000 rg/ml GH-B18 = 2.0 IU. No significant differences (x2) have been found between GH-B18-treated and control cultures, although the highest concentration of GH-B18 has given rise to a few lens vesicles. Differences were not significant. See Fig. 1 for further explanation of the histograms.

A bovine thyrotropin preparation pure enough for amino acid sequencing has been reported to have a biological activity of 30 to 40 IU/mg (Pierce et al., 1973), whereas the preparation TSH-B8 has an activity of 3.5 IU/mg (Reichert and Wilhelmi, 1978). Thus, the active thyrotropin content of TSH-BS may have been about 10% by weight. When the variation of lens regeneration stages in control cultures is considered, all of the tested thyrotropin TSH-B8 concentrations from 3000 to 30 pg/ml seemed to stimulate lens regeneration equally well. From this it appears possible that lens regeneration is stimulated by thyrotropin itself. However, it is still possible that other contaminating compounds in this hormone preparation may be responsible for this

effect. A myoblast growth factor has been reported to contaminate bovine thyrotropin preparations (Gospodarowicz et al., 1975), and the mitotic action exerted by high concentrations of thyrotropin TSH-B5 or B6 preparations, at the lowest 69.5 pug/ml, has been attributed to an impurity (Malemud and Sokoloff, 1974). Although TSH-B8 has been purified by various chromatographic procedures and dialysis, and residual lutropin activity has selectively been diminished by peroxidation (Reichert, 1975), some growth-promoting activity different from thyrotropin may still remain in it. Some stimulation of lens regeneration was observed in irises cultured with high concentrations of follitropin FSH-Bl and somatotropin GH-B18. Thyrotropin is CONTROLS

PRL-a4 ,r,se;;g,es2

/

3.

4,

stages 5 ~ 6,

1

a ,

7,

213

4

5

6

7

8

irlses'm la

q

,

.

1

.

11600

"g/ml

15

4

FIG. 13. Lens regeneration stages achieved by dorsal irises after 20 days of culture with or without bovine prolactin 1600 pg/ml PRL-B4 = 29 III. Differences were not significant. See Fig. 1 for further explanation of the histograms.

1

preparation

PRL-B4;

Pituitary

CUNY AND ZALIK

Hormones

CONTROLS

ACTH-6002

stages 12

3

4

5

stages 6

7

i

8

41

2

3

4

5

6

7

a

irises/

I

irisest

16

33

in Lens Regeneration

15

3 ug/ml

FIG. 14. Lens regeneration stages achieved by dorsal irises after 20 days of culture with or without porcine adrenocorticotropin preparation ACTH-6002; 300 rig/ml ACTH-6002 = 26 IU. Differences were not significant. See Fig. 1 for further explanation of the histograms.

present in these preparations as a contaminant (0.02 and 0.05 IU/mg, respectively), and may account for this effect. Even though thyrotropin was present in lutropin LH-BlO (0.17 IU/mg) this preparation seemed to inhibit the activation of dorsal irises. This apparent effect of LH-BlO needs to be studied further. After explantation, cultured dorsal irises will start to wrinkle and gradually round up into a ball with the stroma on the inside and the iris epithelium exposed to the outside. Also, in some areas tissue edges may coalesce. This process takes roughly 10 to 15 days. Frequently, the lens fiber cells formed on the region of the lens regenerate which was directly exposed to the culture medium, whereas depigmented cells, resembling lens epithelial cells, frequently occupied the portion of the lens facing the inside of the iris tissue ball (Figs. 2b, 7a, 8a). Lens regenerates which were completely enclosed by other iris cells on the inside of the tissue PITUITARY

GLAND

CONTROLS

stages 12

3

4

ball often did not reach a higher developmental stage, or had an abundance of lens epithelium-like cells (Fig. 3). This situation is reminiscent of the one found in vivo in which lens fibers always develop on the side closest to the presumed retinal source of lens stimulators (LeCron, 1907; Coulombre and Coulombre, 1963; Reyer, 1977). Cultured lens regenerates, compared to those in vivo, show different degrees of abnormalities. Morphological aberrations have also been found in lens regenerates arising from irises cultured with pituitary glands (Connelly et a.Z.,1973) and larval frog retinas (Yamada et aZ., 1973), and can also be observed in vivo in lenses regenerating from iris grafts in the eye (Wachs, 1914; Reyer, 1956). These morphological deficiencies can be expected in the culture situation where the natural morphogenetic fields cannot be correctly simulated. The presence of y-crystallin has been used as an in-

5

6

7

stages

a

12

3

4

5

6

7

8

I irisesl

I

irises 12 l **

15

FIG. 15. Lens regeneration stages achieved by dorsal irises after 20 days of culture with or without a newt pituitary gland in close apposition. The frequency of specific lens regeneration stages (stages 3 to 8) is significantly higher in cultures supplemented with pituitary glands as compared to control cultures. The significance levels (x2) are: ***P < 0.005 and “P < 0.10 (almost significant). See Fig. 1 for further explanation of the histograms.

34

DEVELOPMENTALBIOLOGY

dication of lens fiber differentiation (Yamada et al., 1973; Connelly, 1977b). During this study, y-crystallin was detected by immunofluorescence in all fiber cells which could be recognized morphologically and by their eosinophilic staining reaction. In the presence of TSHB8 or newt pituitary gland, some cultured irises in which a regenerated lens was not visible contained a population of often scattered depigmented cells which were positive for y-crystallin. This suggests that even where no lens was morphologically distinguishable, some cell-type transformation had taken place. At this point it is not possible to confirm or deny whether thyrotropin itself is the compound that stimulates lens regeneration or whether an impurity in the preparation used here is responsible for this effect. It is intriguing that the pituitary gland, a tissue not necessary for lens regeneration (Nikitenko, 1940), can nevertheless produce a lens regeneration-stimulating substance. It is conceivable that, after lentectomy, the retina itself can produce a thyrotropin-like compound that induces lens regeneration. Tissues other than the pituitary gland have been reported to contain thyrotropin-like activities at concentrations higher than those present in the serum, such as the human placenta (Hershman and Starnes, 1971), the amygdaloid nucleus in the rat brain (Krieger and Liotta, 1979), and the hypothalamus (Moldow and Yalow, 1978). It is also possible that thyrotropin is only one of several compounds which are capable of stimulating lens regeneration in the newt. We thank Dr. Richard E. Peter for his valuable suggestions concerning pituitary hormones, Dr. Saul Zalik, Ms. Anne Campbell, and Dr. Frank Hampel for their advice in statistical matters. The bovine pituitary hormone preparations were a generous gift from the II. S. National Institute of Arthritis, Metabolism and Digestive Diseases. This work was supported by the Social Sciences and Humanities Research Council of Canada (RC), the Fund for the Support of Young Scientists, University of Basel, Switzerland, Janggen Piihn’s Foundation, Switzerland (RC), the Natural Sciences and Engineering Research Council, and the National Cancer Institute of Canada. REFERENCES ARRUTI, C., and COURTOIS,Y. (1978). Morphological changes and growth stimulation of bovine epithelial lens cells by a retinal extract in vitro. Exp. Cell Res. 117, 283-292. BEEBE, D. C., FEAGANS,D. E., and JEBENS,H. A. H. (1980). Lentropin: A factor in vitreous humor which promotes lens fiber cell differentiation. Proc. Nat. Acad. Sci. USA 77. 490-493. BONNET,C. (1781). Sur les reproductions des salamandres. In “Oeuvres d’histoire naturelle et de philosophie” (S. Fauche, ed.), Vol. 2, pp. 175-179 Neuchltel. COLUCCI,V. L. (1891). Sulla rigenerazione parziale dell’occhio nei Tritoni. Istogenesi e sviluppo. Mem. R. Accad Sci. 1st. Bologna 5, 593-629.

CONNELLY,T. G. (1977a). Influence of indomethacin on lens regen-

VOLUME81, 1981 eration in the newt Notophthalmus

viridescens.

Wilhelm

Roux

Arch. 181, 103-106.

CONNELLY,T. G. (1977b). Pituitary enhancement of Wolffian lens regeneration in vitro. Spatial and temporal requirements. J. Exp. 2001. 200, 359-364.

CONNELLY,T. G. (1977c). Lens regeneration in vitro: Effect of hormones. Amer. 2001. 17, 905. CONNELLY,T. G., ORTIZ,J. R., and YAMADA, T. (1973). Influence of the pituitary on Wolffian lens regeneration. Develop. Biol. 31,301-315. COULOMBRE, J. L., and COULOMBRE,A. J. (1963). Fiber elongation and lens orientation. Science 142. 1489-1490. EGUCHI, G., ABE, S., and WATANABE, K. (1974). Differentiation of lens-like structures from newt iris epithelial cells in vitro. Proc. Nat. Acad. Sci. USA 71, 5052-5056. EISENBERG,S. E., and YAMADA, T. (1966). A study of DNA synthesis during the transformation of the iris into lens in the lentectomized newt. J. Exp. 2001. 162, 353-367. GOSPODAROWICZ, D., WESEMAN,J., and MORAN, J. (1975). Presence in brain of a mitogenic agent promoting proliferation of myoblasts in low density culture. Nature (London) 256, 216-219. HASEGAWA, M. (1965). Restitution of the eye ?rom the iris after removal of the retina and lens together with the eye-coats in the newt-Tritu+us

pyrrhogaster.

Embryologia.

8, 362-386.

HERSHMAN,J. M., and STARNES,W. R. (1971). Placental content and characterization of human chorionic thyrotropin. J. Clin. Endocri,nol. Metab. 32, 52-58.

HORSTMAN,L. P., and ZALIK, S. E. (1974). Growth of newt iris epithelial cells in vitro. A study of the cell cycle. Exp. Cell Res. 84, 1-14. KRIEGER,D. T., and LIOTTA, A. S. (1979). Pituitary hormones in brain: Where, how, and why? Science 205.366-372. LE CRON,W. L. (1907). Experiments on the origin and differentiation of the lens in Amblystoma.. Am.er. J. Anat. 6, 245-257. MALEMUD,C. J., and SOKOLOFF,L. (1974). Some biological characteristics of a pituitary growth factor (CGF) for cultured lapine articular chondrocytes. J. Cell. Physiol. 84, 171-180. MCDEVITT. D. S., MEZA, I., and YAMADA, T. (1969). Immunofluorescence localization of the crystallins in amphibian lens development, with special reference to the y-crystallins. Develop. Biol. 19, 581-607. MILSTONE,L. M., and PIATIGORSKY,J. (1977). &Crystallin gene expression in embryonic chick lens epithelia cultured in the presence of insulin. Exp. Cell Res. 105, 9-14. MOLDOW,R. L., and YALOW, R. S. (1978). Extrahypophyseal distribution of thyrotropin as a function of brain size. Lzfe Sci. 22, 1859-1864. NIKITENKO, M. F. (1940). On the role of the hypophysis in the restoration of the crystallin lens. By&l. Eksp. Biol. Med. 9, 211-214. N~THIGER, R., MCDEVITT, D. S., and YAMADA, T. (1971). Comparison of y-crystallins from frog and newt. Exp. Eye Res. 12, 94-98. PIATIGORSKY,J. (1973). Insulin initiation of lens fiber differentiation in culture: Elongation of embryonic lens epithelial cells. Develop. Biol. 30, 214-216. PIERCE,J. G., LIAO, T.-H., and CARLSEN,R. B. (1973). The chemistry of pituitary thyrotropin. In “Hormonal Proteins and Peptides” (C. H. Li, ed.), Vol. 1, pp. 17-57. Academic Press, New York. POWELL,J. A., and SEGIL,N. (1976). Secondary lens formation caused by implantation of pituitary into the eyes of the newt, Notophthalmus. Develop. Biol. 52, 128-140. RABL, C. (1898). Uber den Bau und die Entwicklung der Linse. I. Selachier und Amphibien. 2. 2001. 63, 496-572. REDDAN,J. R., and WILSON,D. C. (1978). Insulin growth factor triggers mitosis in the epithelium of mammalian lenses cultured in a serum free medium. J. Cell Biol. 79, (2 Pt. 2). 67a.

CUNY AND ZALIK

Pituitary

REICHERT, L. E. (1975). Purification of anterior pituitary hormones (ovine, bovine, rat, rabbit). In “Methods in Enzymology” (B. W. O’Malley, and J. G. Hardman, eds.), Vol. 37, Part B, pp. 360-380. Academic Press. New York. REICHERT, L. E., and WILHELMI. A. E. (1978). Biological activities of recent preparations of pituitary hormones produced under the program of the National Institute of Arthritis, Metabolic and Digestive Diseases. Endocrinology 102. 982-983. REYER, R. W. (1956). Lens regeneration from homoplastic and heteroplastic implants of dorsal iris into the eye chamber of Triturus viridescens and Ambystoma punctatum. J Esp. Zool. 133, 145-190. REYER, R. W. (1971). DNA synthesis and the incorporation of labelled iris cells into the lens during lens regeneration in adult newts.

Develop. Biol. 24, 533-558. REYER, R. W. (1977). Repolarization of reversed regenerating lenses in adult newts, Notophthalmus vi,ridescens. Exp. Eye Res. 24, 501-510. REYER, R. W. (1977). The amphibian eye: Development and regeneration. In “Handbook of Sensory Physiology” (F. Crescitelli, ed.), Vol. 2(5), pp. 309-390. Springer Verlag, Berlin. REYER, R. W.. WOOLFITT. R. A., and WITHERSTY. L. T. (1973). Stimulation of lens regeneration from the newt dorsal iris when implanted into the blastema of the regenerating limb. Develop. Biol. 32,258-281. ROTHSTEIN, H., VAN BUSKIRK, R., and REDDAN. J. (1976). Hypophysectomy inhibits hyperplasia in the adult frog lens. Ophthalmol.

Res. 8, 43-54. ROTHSTEIN, H., and WORGUL, B. V. (1973). Mitotic variation in the lens epithelium of the frog. II. Possible role of temperature and hormones. Ophthalmol. Res. 5, 151-167. SATO, T. (1940). Vergleichende Studien iiber die Geschwindigkeit der Wolff’schen Linsenregeneration bei Triton taeniatus und bei Diemyctylus pyrrhogaster. Wilhelm Roux Arch. 140. 570-613.

Hormon.es in Lens Regeneration

35

SPEMANN, H. (1905). Uber Linsenbildung nach experimenteller Entfernung der primaren Linsenbildungszellen. Zoo/. Arlz. 28,419-432. STONE, L. S. (1957). Regeneration of iris and lens in hypophysectomized adult newts. J. E.rp. Zool. 136, 17-34. STONE, L. S., and STEINITZ, H. (1953). Effects of hypophysectomy and thyroidectomy on lens and retina regeneration in the adult newt, Triturus v. viridescens. J. Exp. Zool. 124, 469-504. TAKANO, K. (1962). On the role of the hypophysis in lens regeneration in Triturus pyrrhogaster. Mie Med. J. 11, -199-508. TAI