Steroidogenesis by enzyme-dispersed turtle (Chrysemys picta) ovarian cells in response to ovine gonadotropins (FSH and LH)

Steroidogenesis

by Enzyme-Dispersed Turtle (Chrysemys Ovarian Cells in Response to Ovine Gonadotropins (FSH and LH)’

picta)

Follicular cells obtained by collagenase dispersion of turtle (Chr~st,r~~s @tr 1 ovarian tissue at three different times of the year (summer. stage I ; fall. stage 2: spring, stage 3) were examined for steroid (progesterone. te\tusterone. estradiol) secretion in response to increasing concentrations of ovine FSH and 1-H. At all three stages of the cycle FSH stimulated steroid production. Although both progesterone and testosterone were secreted by cell\ obtained from ovaries at stages I and 1 of the cycle. estradiol could not be detected in incubations of cells at these times. However, at stage 3 of the cycle, a stimulatory effect of FSH on estradiol production was apparent. The slight stimulatory effect of high doses of LH on progesterone and testosterone production may be due to FSH contamination in this particular NIH preparation. Turtle luteal cells prepared by the same method secreted progesterone in response to dihutyryl iyclic AMP.

Classical studies established that in reptiles, as in atl other vertebrate groups, gonadal function is dependent upon the pituitary gland, and that gonadal function could be largely restored in hypophysectomized animals by injections of mammalian gonadotropins or homologous pituitary extracts (see reviews by Miller. 1959; Dodd, 1960). Despite significant progress since this early work, the nature and biological function of reptilian gonadotropins remains poorly understood. Two protein fractions chemically similar to mammalian LH and FSH have been identified in turtle pituitary extracts (Licht and Papkoff, 1974), but the biological role of each is yet to be clearly defined. In mammals. LH is known to stimulate steroidogenesis in vitro (Savard rf tri.. 1965), and three separate studies have demonstrated a similar in \‘itro stimulatory effect of ovine LH, but not FSH, on the incorporation of radioactive precursors into ’ Supported Ian P. Callard.

by NSF

0016-648Oi78/0343-0304$0 Copyright All rIghI\

Grant

No.

I .0010

G 197X by Acrdemx Press. Inc. of rcproductl”” I” ,A,,) form ,ewrved

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steroid hormones in incubations of minced ovarian tissue from both turtle and snake (Chan and Callard, 1974; Callard et al., 1976; Lance and Lofts, 1977). In contrast, many in \GJO studies have shown that mammahan FSH, but not LH, could support spermatogenesis and steroiddependent secondary sex characteristics in hypophysectomized male lizards and ovarian growth and ovulation, vitellogegesis, and oviduct growth in hypophysectomized female lizards (see review by Licht, 1974). Other studies have demonstrated that, although mammalian FSH appears to be the more potent hormone. LH has a small but significant effect in hypophysectomized lizards (Eyeson, 1971: Burns and Richards. 1974; Licht and Papkoff, 1973).This picture is further complicated by some recent data which showed that both LH and FSH fractions from a wide variety of species, including reptiles, were able to stimulate progesterone production (as measured by radioimmunoassay) from endsgenous precursors in turtle and caiman ovarian frag-

STEROIDOGENESIS

ments in vitro (Crews and Licht, 1975; Licht and Crews, 1976). Although it appeared that, in contrast to studies in which radioactive precursors were used, FSH of either mammalian or reptilian origin was the more potent hormone in eliciting progesterone production, the results were extremely variable, and in some cases LH appeared to give a greater response than that of FSH. The recently developed cell dispersion technique has been used with considerable success in the study of endocrine function in a wide variety of mammalian tissues including pituitary (Portanova et al., 1970), testis (Catt and Dufau, 1973) ovary (Bajpai et ul., 1974) and adrenal (Swallow and Sayers, 1969). Trypsin-dispersed interrenal cells have been successfully used to study ACTH action in the turtle (Callard, 1975). In order to evaluate further -the action of mammalian gonadotropins in the turtle and to rule out the extreme variability encountered in studies using reptilian ovarian fragments in vitro, we have attempted to apply this technique to the study of the effects of gonadotropins on steroid production in the turtle ovary. MATERIALS

AND

METHODS

Animds. Adult female Chr~:ysemys pic’tu. 300-700 g in body weight, 15-17 cm in plastron length, were obtained from a commercial supplier in Wisconsin or were trapped locally. Northern populations of Ckrxsem.vs pi&r produce a single clutch of between four and eight eggs during a period that extends from late May to early July. During the rest of the year the ovary contains yolked follicles in various stages of development. There are two periods of ovarian growth, the first prior to hibernation in the fall. and the second immediately prior to ovulation in the spring (Cagle. 1954: Powell, 1967. Ernst. 1971: Moll and Legler. 1971). We have arbitrarily) divided the ovarian cycle into three stages. Stage I: The cycle begins in late June or July after oviposition has taken place. At this stage. as in subsequent stages. the ovary contains yolked follicles in three size classes: Class I. I2 m m in diameter. follicles that are destined to ovulate the following summer: Class II. 6 m m in diameter: and Class III. less than 2 m m in diameter. In addition to these larger follicles there are many more small yolked follicles less than I m m in diametei

IN

Chrysemys

picttr

305

developing from the germinal epithelium. The ovary remains in this condition until late summer. Stage 2: Beginning in September there is a sudden increase in ovarian growth, Class I follicles increase in size to approximately 16 m m in diameter; Class II, to 8 mm: and Class III, to 6 mm. Between the time the animals enter hibernation in late fall and the time they emerge in early spring, no significant changes in follicular diameter or ovarian weight occur. Stage 3: During April there is a second increase in ovarian growth prior to ovulation. Class I follicles are now I8 m m in diameter and reach a preovulatory size of 20 mm: Class II, I2 mm: and Class III, 6 mm. After ovulation and during the time the eggs are in the oviduct (2 to 3 weeks) the ovary contains corpora lutea. This might be termed Stage 4. but these structures rapidly regress once the eggs have been laid. Preliminary experiments (data not presented) to establish optimum conditions for the cell incubations were carried out in the summer months using tissue from the turtles at Stage 1 of the cycle. Subsequently, a pool of follicular tissue obtained from three to five animals at each of the identified stages of the ovarian cycle was used for each separate experiment. Only one complete experiment was done at Stages I and 3 of the cycle, and three separate experiments were done using tissue from Stage 2. Since the latter all yielded similar results, only one is presented here. Ce// dispersion. The method described below is essentially that of Bajpai et ul. (1974) with a few minor modifications. Turtles were killed by decapitation and a blood sample was collected from the neck for subsequent steroid assays. The plastron was removed, the ovaries were excised and weighed, and the number and size of the yolked follicles were determined. In each experiment only the largest (Class I) yolked follicles were used. The follicles were carefully dissected free from the ovary, adherent connective tissue and blood vessels were removed, and the yolk was carefully extruded. The tissue was then washed several times in Krebs-Ringer bicarbonate glucose (KRBG), pH 7.4, at 32”. blotted, weighed, cut into small pieces with scissors, and placed in a 20-ml glass vial containing IO ml of collagenase solution (Type I CLS, Worthington: 400 units/ml) in KRBG. The vial was then flushed with 95% 0,/S% CO,, capped, and placed in a Dubnoff shaking incubator ( 100 oscillations/min) the undigested the supematant

at 32”. After I hr the vial was removed, fragments were allowed to settle, and containing free cells was decanted into

a SO-ml polypropylene centrifuge tube through a nylon mesh (ca. 80-pm mesh). Another IO ml of collagenase solution was added to the remaining fragments which were then broken up by gently withdrawing into and expelling the solution from a I-ml glass tuberculin syringe without aneedle. The incubation was continued for another hour and the remaining cells were collected in the same centrifuge tube as was the first portion. The

volume in the centrifuge tube was made up to40 ml with KRBG and the cells were centrifuged fbr 15 min at 600~. The centrifuge was nraintained at room temper;t;i~i t‘ The cells were washed three time5 by resuspending the pellet. after discarding the bupernatant. 111 40 111; TV/ KRBG. After the third centrifugation. cell via&lit\ wak assessed by the dye exclu\lon teht uging t).05~, nigrosine in KRGB (Hank\ and Wallace, 1965: C‘clI number was determined using a hemocytometel. The pellet from the final centrifugation was resuspended in KRBG containing O.S’G bovine serum albumin and the volume was adjusted such that I ml of suspen\Ion medium contained 250,000 cell\. While gently stirring. 0.9-ml aliquots from the pool were dispensed into g:la\s vials and 0. I ml of medium containingo, 100 pg, I ng, IO ng, I wg,or 10~gofeitherovineLH(NIH-Sl9)orovlnc FSH (NIH-SI I) was added to each vial. Each hormone concentration was run in triplicate, and six control \i;rls lacking hormone wet-e randomly distributed throughout the incubation. In the experiment in which cells from follicles at Stage 2 of the cycle were used, additional vials were run to which I pg of FSH plu\ I gg of i>H were added: and in the experiment using cells from Stage I of the cycle. an additional serie\ of LI~I\ uert’ run to test the gonadotropic potency of I and 10 ~g L\et wt of homologous pituitary extract. Progesterone (P) and testosterone (‘1‘) were assayed as the end point\ in the bials to which pituitary extract was added. ,411 \ iill\ were flushed with 9% 0,/S? (‘Oi. capped. and pla~.c~l in a Dubnoff shaking Incubator- of 31” for 3 hl- ‘~1 10 oscillation\imin. 4t the end of the incubation period the vials were frozen and stored at IS” until anal)reci. A single experiment was performed using dispel-Ted ccII\ from postovulatory fotliclez (corpora lutea) of 1u1tit.s with eggs in the oviducts. Dispersion techniques ,md incubation conditions were identical to thu\e 01 the experiments in which follicular tissue was u\ed. but the final concentration of cell\ incubated was only 40.000, ml. To each triplicate \et of vials were added 0. I, I .O. 2.0, and 10 m M of N’,.
RESULTS Optimum enzymic dispersion was ohtained when approximately 1 g of tissue; IO ml of collagenase was used. Yields from collagenase-digested follicuiar tissue \\er’e low (between 5 and IO i 10” cells/g:wet weight of tissue), but viability wa\ consistently above 85%. 4 heterogeneous population of cells ranging in sire from approhimately 7 to 17 km was obtained. and it was not possible to determine uhether thc\e cells were of thecal or granulosai origin. Cells remained viable for up to 12 hr a4 assessed by the dye exclusion test. hltt steroid hormone concentration in the medium reached a maximum at 3 hr and did not change appreciably if the incubation w;ts prolonged.

Progesterone and testosterone were detectable in the incubation media in all experiments. In incubates of cells from Stage 1 follicles the addition of ovine FSH to the medium caused a slight increase in P production at the IOO-ngiml dose and a maximal stimulation at the I-pg/ml dose. No further increase in P production was elicited at the highest dose of FSH tested (10 pgiml). Similar dose-response curves were obtained with cells from ovaries at Stages 2 and 3 when incubated in the presence of FSH, but the maximum P concentration3 reached were smaller than those recorded in incubates from Stage 1 follicles (Fig. 1). No differences in T production were detected at any dose of FSH in cells from Stage 1 ovaries. In incubates of cells from Stage 2 follicles, however. a response to a lOO-rig/ml dose of FSH was apparent, but no further increase in T was obtained ufith either the 1- or IO-pg/ml dose of FSH. The concentration of T reached in response to

STEROIDOGENESIS

307

IN Chrys~mys pictu

stage 3

/

J

&

Y

100 ,

’ 0

I

I

1

I

lwl

long

ioong

lug

FSH/ml

added

I 1oug

FIG. 1. Progesterone production by enzymically dispersed ovarian follicular cells from Chrysemys pictu, at three different stages in the annual ovarian cycle, incubated in the presence of FSH. The solid circles represent the mean k SE values obtained using tissue from stage 1 of the cycle (July-October); the open squares, stage 2 of the cycle (NovemberDecember); and the solid triangles, stage 3 (AprilMay). Progesterone values are expressed as picograms per milliliter of incubation media.

FSH by cells from Stage 3 ovaries (4000 pgml) was approximately 50 times greater than that obtained in incubates of cells from Stage 2 ovaries (Fig. 2). The addition of LH to the incubates of Stage 1 cells did not cause any significant increase in P concentration at the lower doses tested but did cause a significant increase at the IO-pg dose. No effect of LH was detected in incubates of Stage 2 follicle cells, and only a slight increase at the IO-pg dose in the Stage 3 incubates (Fig. 3). The production of T was not stimulated by the addition of LH to the cells from ovarian Stages 1 and 2, but did cause a significant increase in T at the highest dose tested in cells from Stage 3 (Fig. 4). Estradiol could not be detected in any of the incubations of cells taken from ovaries at Stages 1 and 2 of the cycle regardless of the dose of gonadotropin added, but was detectable in incubates of cells from Stage 3 follicles in the absence of gonadotropin. Addition of FSH to Stage 3 incubates caused a significant increase in E at the I-,ug dose but not at the lOO-ng dose. LH

i? e 8 ti

:

F

I 0

1 1w

1 long FSH/ml

1 loong

I lug

4 1o.w

added

FIG. 2. Testosterone production by follicular cells in the presence of FSH. Symbols as in Fig. 1.

had no detectable effect on E production in these incubations (Table 1). P and T production in the vials to which 1 pg of FSH plus 1 pg of LH were added at Stage 2 of the cycle were no different from the vials which contained 1 pg of FSH alone. The addition of the equivalent of 1 pg wet weight of fresh turtle pituitary extract at

P stage 1

stage 2

& I

t 0

stage 3 i

I

L

lng

long

1 loong

1 lug

i 1oug

LHlmladded

FIG. 3. Progesterone production by follicular cells at three stages of the cycle in the presence of LH. Symbols as in Fig. 1.

Gonadotropin

I .H

FSJ-4

without effect (Table 3). T and E were not measured in these incubations.

LH /ml added

FIG. 4. Testosterone in the presence of LH.

dose

production by follicular Symbols as in Fig. I.

cells

Stage 1 of the cycle caused a significant increase in P production (801 2 205 vs 268 f 10 pg/ml for controi), and IO pg of pituitary extract resulted in a P concentration (1668 or.402 pg/ml) not significantly different from that produced by the addition of 1 pg of ovine FSH. T production was also increased slightly, but not significantly, by the addition of IO pg of pituitary extract. Injhrnce of DiBu-CAMP on P prodrrction by Turtlr Lutrirl Cells The addition of I rn&I DiBu-cAMP to suspensions of luteal cells significantly increased P production over that seen in control vials. No further increases in P were obtained at increaseddoses of DiBu-CAMP. and the lowest dose tested, 0.1 r-r&I, was

DlSCUSStON The results of these experiments demonstrate that ovine FSH is much more active than ovine LH in stimulating steroid production from endogenousprecursors by enzymicalty dispersed turtle follicutar cells. The responseto FSH in terms of P production is much greater than the response in terms of T production at Stages I and 2 of the cycle: however, at Stage 3 (just prior to ovulation), the secretion of T in responseto FSH is much greater than P secretion. O n ly cells from Stage 3 follicles produced E, and E production was increased in the presence of FSH. These results for ovine FSH are in agreement with similar studies using ovarian fragments of other turtle species (Crews and Licht. 1975; Licht and Crews, 1976) in which P and E were measured. Crude turtle pituitary homogenate was much more potent than the purified ovine

2 TABLE INFLUENCE OF DiBu-CAMP ON P PRODUCTION ~%RTL.E LUTEAL CLLLS IN VITRO

BY

0 P concentration (w/ml) ” Mean

2 SEM.

292 2 59’1

294 i

II4

1042 i

235

II42

r 352

1042 + 236 -I__

STEROIDOGENESIS

FSH in stimulating P production, 10 pg wet wt of fresh pituitary being equivalent in stimulatory activity to 1 pg of ovine FSH. It should be noted that FSH and LH fractions of chelonian origin (Licht and Papkoff, 1974) were similar in steroidogenic activity in the in vitro ovarian system used by Crews and Licht (1975) and Licht and Crews (1976). In vitro isotopic studies have provided strikingly different results, since ovine LH, but not FSH, has been repeatedly demonstrated to be most active in stimulating steroidogenesis in Chrysemys (Ghan and Callard, 1974; Callard er ul., 1976) and in the snake Nuju nuja (Lance and Lofts, 1977). The basis for this apparent contradiction remains to be elucidated in the reptile, but Oakey and Stitch (1968) and Mori (1975) explained gonadotropin-induced decreases in [4-“YJtestosterone incorporation into estrogens in mammalian ovarian incubations by suggesting that the pool of endogenous T was enhanced by the hormonal treatment, thus leading to the apparent reduction in radiolabel incorporation. Steroidogenesis in reptilian ovarian tissue (corpus luteum) is stimulated by DiBuCAMP in the range found effective in mammals (Marsh, 1976). This would suggest that gonadotropins act in a similar manner in reptiles and mammals. The steroidogenic ability of the luteal cells taken from the turtle appeared to be far greater than that of the follicular cells since only 40,000 cells/ml produced an amount of P equal to that produced by 250,000 follicular cells. This result however, could have been due to a purer cell preparation obtained from the corpora lutea which are a solid mass of homogeneous cells, whereas the follicular wall of the preovulatory oocyte is composed of several different types of cells, many of which appear to be typical fibrocytes. The corpora lutea of reptiles, however, are believed to be a rich source of P, since several studies have shown that plasma level:, of P are highest in pregnant

IN Chrysemys pictu

309

viviparous reptiles which have corpora lutea present in the ovary (Chan et al, 1973; Highfill and Mead, 1975). The results of this study suggest that there is a seasonal variation in the steroidogenic response of turtle follicular cells to FSH, probably reflecting changes in tissue content of steroidogenic enzymes and/or changes in gonadotropin receptors. When T production (or T and E) is used as the end point, maximum changes in tissue sensitivity to FSH occur between the hibernation period and ovulation. In contrast, P production was maximally stimulated in the postovulatory period. Caution must be exercised in the interpretation of such seasonal observations however, since only a few experiments were conducted at each stage of the ovarian cycle, and interassay variations may contribute to the observed differences. However, plasma levels of E and T in the turtle are highest during this phase of the annual ovarian cycle (Callard rr al., in press), suggesting that the preovulatory follicles are responding to endogenous gonadotropins at this time and that the seasonal changes in the sensitivity of the dispersed cells to ovine FSH probably correspond to a seasonal change in sensitivity occurring in nature. Throughout these studies, a stimulatory effect of ovine LH on steroid production was noted at the higher dose levels. This effect may be accounted for on the basis of FSH contamination of the LH. Ovine LH S-19 contains 0.05 FSH S-l units/mg; (5%); 10 ,ug of LH-S-19 therefore contains about 500 ng of FSH. It can be seen by inspection of the figures that the stimulatory effect of 10 pg of LH is approximately equal to that expected of 500 ng of ovine FSH, suggesting that the stimulatory effect of high doses of LH seen in these experiments are probably due to FSH contamination. However, since in other in vitro systems LH is stimulatory where FSH is not (Chan and Callard, 1974; McChesney et al., 1976) it is

possible that ILH may have a stimtllatory action on steroid biosynthesis in the ahsence of FSH. Indeed, l,icht and Pupkofl (1973) have demonstrated that ovine l>H has intrinsic activity in hypophysectomized male lizards when spermatogenesis and growth of epididymis and renal sex segment were measured as indexes of gonadotropic stimulation. Since only the large Class III follicles were reported here. the lack of LH activity may be due to developmental changes in either the amount or type of gonadotropin receptor present in the granulosa cells. This is supported by the fact that clear-cut actions of LH on ovarian steroid secretion in r*iv/j are observed when the hormone is injected at one time of the year, but not at another (Callard and Lance. 1977). In the latter situation, the total ovarian response is evaluated, and thus different classes of follicles may contribute. Studies of the actions of mammalian gonadotropins in other vertebrate phyla make an interesting comparison to the Reptilia since, in teleosts (Burzawa-Gerard and Fontaine, 1972),amphibia (Licht and Crews. 1976;Muller, 1976),birds (Maung, 1976).and mammals (Savard rt ul., 196S), LH appears to be the most active steroidogenic hormone. However, evidence is accumulating that FSH may have a specific action on estrogen synthesis in the rodent gonad (Dorrington rt ul., 1975). At present it seems fair to say that at least one of the two hormones can activate steroidogenesis in all vertebrates, but that some confusion exists due to the difficulty of defining the biological activity of the putative nonmammalian gonadotropins using an array of heterologous bioassays. It has been observed by Wallis (1975) that only minor changes in the amino acid sequences of pituitary hormones have occurred during evolution, and it is unlikely that the structures of reptilian gonadotropins are radically different from those of other vertebrate groups, since they show both immunological (Licht et crl., 1974) and chemical

similarit& (Licht of (ii.. 1976). 19,would 4eem. therefore, that in addrtion to \tudiex on the nature of the gonadotropin~, considcration 01’ the evc,lution \,f
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Licht, P., Papkoff, H., Goldman, B. D., Follett, B. K., and Scanes. C. G. (1974). Immunological relatedness among reptilian, avian and mammalian pituitary luteinizing hormones. Cert. Comp. Endocrinol. 24, 168-176. Louvet, J. P., Harman, S. M., Schreiber, J. R., and Ross, G. T. (1975). Evidence for a role of androgens in follicular maturation. Endocrinology 97, 366-372. Marsh, J. M. (1976). The role of cyclic AMP in gonadal steroidogenesis. Biol. Reprod. 14, 30-53. Maung, Z. W. (1976). Effect of LH and CAMP on steroidogenesis in interstitial cells isolated from the testis of the Japanese quail. Gen. Comp. Endocrinol. 29, 254 (abstract No. 37). Miller, M. R. (1959). The endocrine basis for reproductive adaptations in reptiles. In A. Gorbman (ed.) “Symposium on Comparative Endocrinology” (A. Gorbman, ed.), pp. 499-516. Wiley, New York. Mall, E. O., and Legler, J. M. (1971). The life history of the neotropical slider turtle, Pseudemys scripta (Schoepff), in Panama. Bull. Los Angeles Co. Mus. Nat. Hist. 11, I-102. Mori, T. (1975). Steroid formation in bovine ovarian follicles. Endocrinol. lapon. 22, 361-366. Muller, C. H. (1976). Gonadotropin regulation of the bullfrog testis. Amer. Zool. 16, 259 (abstract No. 439). Oakey, R. E., and Stitch, S. R. (1968). Oestrogen biosynthesis by segments of bovine follicle and the effect of follicle stimulating hormone preparations. Acta Endocrinol. 58, 407-418. Portanova, R., Smith, D. K., and Sayers, G. (1970). A trypsin technic for the preparation of isolated rat anterior pituitary cells. Proc. Sot. E.rp. Biol. Med. 133, 573-576.

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