Effects of mammalian gonadotropins (FSH and LH) on the testes of the lizard Anolis carolinensis

GENERAL

AN3

COMPARATIVE

Effects

13, 367-381

ENDOCRINOLOGY

of Mammalian Testes PAUL

Department

Gonadotropins

of the LIGHT

of Zoology,

(1969)

Lizard AND

University

Received

(FSH and

Anolis ANITA

carolinensis

K. PEARSON

of California,

March

LH) on t

Berkeley,

California

94720

12, 1969

Responses of the testis to mammalian FSH and LH were examined in the lizard Anolis caPolinen& at different times of year and at different temperatures. Small amounts of FSII (0.1 mu/day) maintain testis weight and the accessory structures (epididymis and sex segment of kidney) and promote the development of new germ cells in surgically hypophysectomized lizards at 31” during the spring. Ovine LH has the same actions but much higher doses are required. With testosterone the rate of posthypophysectomy regression is reduced but no new germ cells develop. Both ovine FSH and LH stimulate spermatogenesis and interstitial cell activity in “physiologically” hypophysectomized Anolis at 31” in the fall. The development of accessory sexual structures requires a higher dose of gonadotropin than does complete spermatogenesis ; only the early stages of spermatogenesis-up to the spermatid stage-occur with low doses of gonadotropin. Comparison of several preparations of ovine gonadotropins (from NIH and Papkoff) indicates that the potency of FS is many hundred times greater than that of LH with regard to the stimulat,ion of spermatogenesis and interstitial cell activity in the lizard. At 29”, the high doses of gonadotropin required to initiate the development of accessory sexual structures cause an abnormal-edematous-response in the germinal epithelium. Kormal spermatogenic recrudescence at 20” is mimicked best by a relatively low dose of gonadotropin which does not promote interstitiai cell activity. The observed thermal effects and differential dose sensitivity of various testicular responses suggest t.hat annual cycles of spermatogenesis and interstitial cell activity in reptiles may result from variations in the level of a single gonadotropin (or gonadotropic-complex) . A scheme is proposed whereby low temperatures suppress circulating gonadotropins so that only the early stages of spermatogenesis occur, while increased, temperatures elevate gonadotropin production which leads to the development of accessory sexual structures and spermiation.

Although the dependence of the testis on the anterior pituitary has been established in reptiles, there is litt,le information regarding the specific actions of the gonadotropins in these vertebrates. Most studies concerning the effects of exogenous pituitary gonadotropins have employed relatively crude extracts or large doses, frequently under undefined environmental conditions (see reviews by Knobil and Sandler, 1963 ; Nalbandov, 1966 ; van Tienhoven, 1968). Consequently, it is not even clear whether reptiles exhibit distinct responses to the two gonadotropins-a fol367

licle stimulating hormone (FSH) and an interstitial cell stimulating hormone (IGSH or LH). The lack of such information hinders interpretation of histological studies which indicate the presence of two discrete gonadotropic cell types in the pars distalis (Grignon and Grignon, 1965 ; St, Girons, 1967). In an attempt to clarify the gonadal-pftuitary relationship in reptiles we sought to determine the sensitivity of the testis to mammalian FSH and LH in the lizar Anolis carolinensis. We measured tbe effects of these hormones on testicular fune-

368

LIGHT

AND

tion in hypophysectomized lizards and on the recrudescence of involuted testes in intact animals. The role of temperature on the response of the reproductive system to exogenous gonadotropins was also examined. MATERIALS

AND

METHODS

Care of Lizards Lizards were obtained from Louisiana. Only adult males with snout-vent lengths over 59 mm were used. Animals were housed in screen-covered aquaria which were kept in controlled-temperature cabinets illuminated by fluorescent lamps (temperatures and photoperiods are indicated below). Hypophysectomized lizards were hand-fed mealworms while others were supplied with mealworms and crickets ad Zibitwm. Water was available at all times. Lizards that lost more than 15% of their initial weight or that appeared to be in poor condition were excluded from final analysis. Assay of Reproductive

Condition

Testicular activity was evaluated by the weight of one testis and by the histological appearancof the seminiferous epithelium (Licht, 1967a). Spermatogenesis was assessed by the proportions of cell types (spermatogonia, primary spermatocytes, early and transforming spermatids, and spermatozoa) in the testicular tubules, and the occurrence of spermiation was determined by the presence of sperm in the epididymis and vas deferens. Quantitative methods for measuring the composition of the seminiferous epithelium are described in Licht and Pearson (1969). Interstitial cells are small and scarce in the testes of Anolis and difficult to measure directly. Consequently, the tubular diameter and epithelial height of the epididymis and sexual segment of the kidney were used as indices of androgenic activity; 10 measurements of each variable were averaged for each individual. The condition of these structures correlates well with changes in the nuclear diameter of interstitial cells in A. carolinensis (Fox, 1958). The dependence of these accessory sexual structures, especially the kidney segment, on androgens seems well established (Prasad and Sanyal, 1967).

PEARSON

ratory of the University of California. The methods of preparation and characteristics of the Papkoff preparations are described in Papkoff (1966) and Papkoff et al. (1965, 1967). A summary of the potencies and levels of contaminations of each preparation used is given in Table 1. Hormone doses are discussed in individual experiments. All gonadotropic hormones were dissolved in distilled water and injected subcutaneously (in the neck region) in 0.02 ml of solution, Injections were made at mid-day. TABLE SUMMARY

1

OF GONADOTROPIN ACTIVITIES AND LH PREPARATIONS TESTED IN Anolis carolinena%

IN

FSH

Mean relative potency (units/mg) b Preparation Exp. 1 NIH-FSH-S4 NIH-LH-Sl 1 Exp. 2-3 NIH-FSH-S6 NIH-LH-S14 Papkoff FSH Papkoff LH

FSH

LH (ICSH)

1.37 O.Ol@

0.017 0.81

1.24 0. 023c 20-30 Nil

0.0045 0.98 ea. 0.0002d 2.0

=Data are based on specifications provided by the Endocrinology Study Section of the NIH for their preparations. Data for Papkoff preparations are based on Papkoff (1966) and Papkoff et al. (1965, 1967), and personal communications. b Units are based on NIH standard preparations: 1 unit is equivalent t,o the activity of 1 mg of NIH-FSH-Sl or NIH-LH-Sl. Conversions to IU are: FSH, 1 unit = 26.6 IU; LH, 1 unit = 1530 IU (Donini et al., 1966). c These values represent maximal activities of FSH contaminations since the largest dose tested failed to give a response. d Values for LH contaminations are highly variable depending on the assay used but the true value may be close to zero (see discussion by Papkoff, 1965, p. 338). Treatment with testosterone (Oreton) consisted of implanting pellets of testosterone weighing 24 mg under the skin on the back.

Hormones

All of the gonadotropins used in this study were of ovine origin. Preparations were obtained from 6he Endocrinology Study Section of The National Institute of Health (NIH) or from Dr. Harold Papkoff of the Hormone Research Labo-

Exp. 1: Maintenance Hypophysectomized

of Testicular Lizards

Functions

in

These experiments were conducted in midMarch when the testes were almost fully enlarged and accessory sexual structures partially devel-

GONADOTROPINS

oped; the epiclidymis was secretory and contained some sperm but the sexual segment of the kidney was only slightly developed (spermatogenie stage 6 of Licht, 1967a). Further studies of the lower doses were made in a second series of animals starting in April; the reproductive system was fully developed in these lizards. The pars distalis was removed (designated as hypophysectomy) under cold anaesthesia and the success of the opera.tion was verified histologically following autopsy (Licht and Pearson, 1969). Temperature was regulated at 31” with a 14-hr photoperiod. Lizards were injected daily with either FSH (NIH-S4), LH (NIH-Sll), or a combination of the two; several doses of each hormone were tested on groups of five to eight animals. Another group was treated with testosterone. Treatment started on the day after hypophysectomy and continued for 14 days. Complete regression of the testes occurs in hypophysectcmized animals in this time (Licht and Pearson, 1969). Autoradiographic studies provided information on the rates of cell maturation. Lizards were injected with 10 pCi of II’-thymidine intraperitoneally 4 days after hypophysectomy.

IN

MALE

LIZARDS

NIH-FSH information

36

daily for 15 days in hopes of obtaining on maximal rates of recrudescence.

Exp. 3: Effects of Temperature sponses to Gonadotropins

on Testicular

Several groups were established to parallel Exp. 2 except that the animals were kept at 20”. Only NIH-FSH and LW were used. Because of the slower response expected at this temperature, animals were autopsied at 30 and 60 days. The results of the above experiment indicated that the doses of hormones used were too high for normal spermatogenesis. A second experiment was subsequently performed in mid-Decem’oer to test the effects of lower doses of FSK at 20”. Because testicular recrudescence had begun by t-hi? time, indicating the presence of endogenous gonadotropins, hypophysectomized animals were used in this test. Rypophysectomized lizards were injected with either 1 or 25 pg FSH(KIH) every ot.her dav for 25 days.

REWJLTS

Exp. 1: Maintenance of Testes in Exp.

2: Testicular

Recrudescence

To test the effectiveness of various gonadotropios in stimulating development of the regressed gonad, we utilized animals during September when the reproductive system is normally quiescent, Testicular regression is normally complete by late August in this species (Fox, 1958; Licht, 196i’a). Previous photo-thermal studies have demonstrated that the production of endogenous gonadotropins can be completely suppressed for about 2-3 months starting in early September if the lizards are kept at 31” with a short (6 hour) daylength (Licht, 1967a,b). High temperature and short days thus effect a “physiological” hypophysectomy with regard to gonadotropins (see also Licht and Pearson, 1969). This method has several advantages over surgical hypophysectomy: It is feasible to use larger numbers of lizards; they require less care and remain healthy for long periods. In these experiments, hormone injections were made every other day. Hormone treatments included several doses of NIH-FSH-S6, NIH-LH514, a combination of the two, Papkoe-FSH, and (this a~koff-~~ treated with neuraminidase treatment inactivates FSH but not LH). At each dose level, six to eight lizards were killed after 15 days (seven injections) and another six to eight lizards were kiiied after 29 days (14 injections). An additional group was injected with 400 pg

Re-

ysectomked

Hy-poyh-

Lizards

Testis weights after 14 days of treatment at 31” in March are summarized in Fig. 1, Survival was good among the controls and FSH-treated groups: but poor among the LH-treated animals at low doses, and only a few of these remained at, the end of the 14 days. Testes of intact controls were significantly larger (p < .02j than in the initial controls sacrificed at the beginning of the experiment; the accessory structures were hypertrophied and the epididymides and yasa deferentia were packed with sperm. Untreated bypophysectomized iizards showed a complete t’esticu!ar regression and involution of the epididymis and sexual segment, of the kidney (Fig. 2). With 0.1 or 1.0 pg of FSH per day, the testes of the hypophysectomized lizards were identical in size and appearanoe La those of the intact controls (Fig. 3). W’ith 100 pg FSH, the testes were about 309 larger than those of intact controls, and this diderence was significant (t < .%?I, The genera! appearance of the semmiferous epithelium, and the hypertrophy of the epididymis and sexual segment of the kid-

370

LIC’HT

AND

PErlRSON

0.5 5 FSHC LH

Hormones

ipg/doy)

FIG. 1. Testicular weights associated with hormonal treatments in hypophysectomiaed Snolis in midMarch when the testes are relatively large (see initial controls) and when the epididymis and sex segment of kidney are enlarged. Ovine hormones (NlH) were given daily for 14 days following hypophysectomy (8). Experimental groups initially consisted of five animals. Vertical bars show mean values for each group and the shaded circles represent individual values.

ney in all FSH-treated lizards was similar to that of the intact controls. In the only two animals surviving in the group receiving 1 ;Lg LH, testes were almost

fully regressed and the accessory structures were involuted. With 10 pg LH, the testes were considerably smaller than they were at the start of the experiment but histolog-

FIGS. 2-3. Photomicrographs for 14 days. Scales show 50 P.

hypophysectomiaed

of testes

from

lizards

FIG. 2. Hypophysectomy without hormone treatment. Tubules spermatogonia and Sertoli cells. FIG. 3. Hypophysectomized lizard injected with 0.1 pg NIH-FSH germinal epithelium is the same as that of intact controls, containing from spermatogonia near the periphery of the tubule to spermatozoa

in mid-March

are involuted

and

and

contain

kept

only

for 14 days. The appearance all stages of spermatogenesis around the lumen.

at 31°

a few of the ranging

GONADOTROPINS

IN

ically these appeared relatively normal; the epididymis and kidney were hypertrophied and sperm were still present in the epididymis. With 100 pg LH, the size and appearance of the testes were equivalent to intact controls (and to those taeated with 0.1-1.0 pg FSH) . No synergism was evident bet,ween FSH and LH; in fact, the combination was less effective in maintaining the testes than comparable doses of FSH alone. For example, the weight of the testes with 0.5 pg FSH -/- 0.5 pg LH was significantly less (p < .O2) than with only 0.1 pg FSH. The testis weights of testosterone-treated animaIs were almost the same as at the start of the experiment, but the seminiferous epit,helium was not normal in appearance. There was a marke.d reduction in the numbers of primary sgermatocytes and spermatids, indicating that, existing cells were evolving but not being replaced. This was verified by autoradiographic data (see below). The accessory sexual structures were highly developed and secretory as expected from androgen treatment, but the abundance of sperm in the epididymis was markedly reduced. Autoradiography confirmed that the gonadotropins were able to promote t.he development of new germ cells in addition to maintaining existing cells. In intact controls injected with thymidine 10 days before autopsy, early spermatids were the most, advanced labeled cell in the testes, and these were numerous. Variable numbers of early spermat.ids were also labeled in ali hypophysectomized FSH- and LHtreated iizards, even in those in which test.icular weight, showed some decline. Thus it appears that’ 10 days represents t#he minim~um time required for the maturation of germ cells to the level of the early spermatid and this rate was unaffected by hormone treatment. There were few labeled cells (mostly spermatogonia) in the testosterone-treated lizards, confirming t,hat testosterone may maintain or allow maturation of existing cells, but it does not support the formation of new spermatocytes. A further test of the minimal effective dose of FSH (NIN) was undert’aken start-

MALE

LIZARDS

371

ing in April. Hypophysectomized animals were given 0.1 or 0.01 &day. Testes in lizards given 0.01 kg FSH did not. differ significantly from those of hypophyseetomized controls. A dose of 0.1 ,ug FSH was less effective in maintaining the testis than it was in the March experiment, but some prevention of regression was still evi.dent. Thus, the minimal effective dose of FS for maintaining the developed t,estis is around 0.1 fig per day. Exp. 2: Testicular Recmdescmce at SPC’ Testis weight. The absence of endogenous gonadotropins in September was confirmed by the fully regressed testes of the control animals t,hroughout the 29-day observation period. The composition of the germinal epithelium in these lizards resembled that of hypophyseetomized animals (cf. Figs. 2 and 6). With all hormone doses, the rate of testicular growt’h (mg,/day) bet,ween O-15 and 15-29 days was essentially the same (Fig. 4). The relation between hormone closesand final testis weights (after 14 injections in 29 clays) illust.rate these data (Fig. 5). The lowest dose of NH-FSH (Ct.1 pg), NIH-LH (1 Pg) and Papkoff FSH (0.01 Fg) failed to elicit a significant increase in t#es”,isweight throughout the 29day period although they had some effect, on spermatogencsis (see below). All higher doses of FSH and LH produced significant (p < 62) testicular growth within 15 days. An analysis of variance for these data (according to Bliss, 1967) indicates tha,t the three highest doses of both NIEET-hormones yielded &sight log dose-response curves, and the curves for FSH and LH were parallel (Fig. 5). The difference in pot,ency between equal weights of the FS and LH is clearly evident from the nosition of the curves. The NH-FSH was a%out 20 times more potent than 214 in promoting testicular growth. The average weight of the testis after 15 daily injections of 400 pg NPH-FSH was 32 mg, but the largest two testes of the eight animals in this group appeared edematous. The weight of the remaining

372

35

LIGHT

-. o--o l

AND

NIH-FSH NIH- LH

Time

(days)

FIG. 4. Effect of mammalian (ovine) gonadotropins on the growth of the testes in Anolis at 31’ starting in the first week of September. Animals were physiologically hypophysectomiaed by high temperatures and short days (6 hr). Injections were given every other day (doses are indicated at right of each curve). Pcints represent averages for six to eight individuals sampled at each time shown.

six averaged 23 mg. This weight is less than that expectec ‘Torn extrapolation of the dose response curve obtained with the 15-day injections of the lower doses of

Dose

PEAR.SON

FSH, indicating that the log-dose-response curve departs from linearity between the 50- and 400~pg doses. We did not test sufficient doses of the Papkoff preparations to obtain detailed dose-response curves, but the results indicate a general parallelism between their slopes and those of the NIH preparations. However, the FSH prepared by Papkoff was about 100 times more potent than the NIH-FSH-or 124 X NIH-FSH-Sl standard (see Table 1). The neuraminidasetreated Papkoff LH was less potent than the NIH-LH-equivalent to about 0.5 X NIH-LH-Sl standard. When FSH and LH (NIH) were given in combination at low doses (1 pg) a slight augmentation of testis weight was observed (Fig. 4). The testes were significantly larger (p = .03) than with 1 pg FSH alone; they were equivalent to that expected with 1.6 pg FSH. In contrast, lizards receiving 10 pg FSH and LH had significantly smaller testes (p < .05) than those receiving 10 pg FSH alone (Fig. 5). Spermatogenesis

The histological appearance with injections of 0.01 and 0.10 FSH were comparable to l- and of NIH-FSH, respectively; and 50-pg doses of Papkoff LH were

( pg /injection

of testes pg Papkoff lo-pg doses the lo- and comparable

)

FIG. 5. Log dose-response curves for the effects of ovine gonadotropins on testis weights in physiologically hypophysectomized Anolis. Animals were injected every other day for 29 days at 31” during September. The mean and 95% confidence limits about the mean for six to eight animals are shown for each dose tested. Regression curves were computed from the values shown (Bliss, 1966).

GONADOTROPTNS

to the same doses of NH-LH. Also, the combined doses of PSH and LH differed little from the same doses of FSH alone. Consequently, discussion will be focused on the effects of the NIH preparations. The relative effects of each hormone on the qualitative and quantitative composition of the seminiferous epithelium resembled those described for testis weights (Figs. 6-12). At the lowest dose of FSH (0.1 pg), there was only a small production of primary spermatocytes; this represents a significant recrudescence in contrast to controls (Fig. 7). With 1 pg FSH there was a pronounced increase in spermatogonia and primary spermatocytes but spermatids were rare (Figs. S-9). The lack of spermatids probably reflects their mort,ality, since pycnotic and phagocytized cells were observed in the t’estes and epididymal canals (Fig. 8) I With increasing doses of FSH, spermatogonial multiplication and primary spermatocyte production increased progressively, but spermatids did not increase proportionally. For example, there was no difference (p > JO) between the average frequency of spermatids with ‘10 and 50 pg FSH and only a slight increase wit,h 400 JL~ FSH, whereas the frequency of spermatogonia and spermatocytes increased significantly with each increase in dose, except at the highest (Fig. 12). The testes of LH-treated lizards contained the same stages of spermatogenesis as the FSH-treated animals at 15 and 29 days. However, the cell numbers in the LN-treat,ed groups were generally less than with 10 yg I?SH, and the degenerating and phagocytized cells were more numerous. The lowest dose of LH (1 pg) had no effect on spermatogenesis. FSH treatment produced a few transforming (tail-stage) spermatids in the initially involuted testes in 15 days, although these do not appear in the testis chosen for illustration (Fig. 10). This rate of germ cell maturation is similar to that observed autoradiographically in the developed testis (see Exp. 1). By 29 days, maturing spermatozoa lined the Iumina of enlarged testicular tubules (Fig. 11) ~However, only

IN

MALE

LIZARDS

373

two of the many animals examined at days showed spermiation-one with t high dose of FSH and one with the big dose of LH. Androgenic

(interstitiai

cell)

Ac~vit~

The relative effe&s of various hormone treatments on adrogenic activity, as judge by the development of the epididymis and sexual segment of ‘the kidney, parallel.ed those observed for testicular growth a.nd spermatogenesis (Fig. 13). FSH was clearly a more effective stimulant than LH, This is evident from both the rates and extent of development of the epididymis and sexual segment of the kidney (Figs. 1617) jl The epididymis responded more rapidly and at lower doses than did the sexual segment of the kidney; in both, an increase in tubular diameter preceded an increase in epithelial height (Fig. 13). After 15 days of t’reatment, the epididymis was maximally hypertrophied with 50 jog FSE fas in Fig. 15). With this dose the sexual segment of the kidney was partly developed at 15 days, and there was a further enlargement in tubular diameter and epitheli height to maximal levels at 29 days. Wi 10 pg FSH, the diameter of the epididym tubules was significantly enlarged wit 15 days but epithelial height did not increase significantly until 29 days (Figs 14-15). There was also a significant change (p < 92) in t,he sexual segment of the kidney after 29 days (as in Fig. 17). Only the high dose (100 pg) or” produced a significant enlargement) of epididymis and it did not reach the dimensions attained with 50 pg FSH. LH did not produce a significant enlargement of the epithelium of the kidney sexual segment even after 29 days, and the diameters of these tubules were on1 (Fig. 13). Thus, 100 pg tive in stimulating an than 10 pg FSH. The O.l-j*g dose of P same effects on the accessory sexual siructures as the lo-pg dose of NIH-IFS There was no significant development epididymis or kidney ith the highest (50 pg) dose of Papkoff L

374

LICHT

AND

PEARSON

Photomicrograph of testes showing the effects of various doses of ovine NIH-FSH in physiFIGS. 6-11. ologically hypophysectomized An&s kept at 31” with 6 hr of light daily in September (refer to Fig. 4). Scales indicate 50 p. AbbreviaGons: spg, spermatogonia; SC-I, spermatocytes I; sptd, spermatids; spz, spermatozoa.

FIG. 6. Uninjected control after 29 days showing involuted FIG. 7. 0.1 ,ug FSH for 29 days; there are some normal degenerating spermatids are evident FIG. 8. 1.0 pg PSII for 15 days; cytes or spermatids are in lumen.

in the lumen. spermatocytes

tubules. appearing

I are abundant,

spermatocytes

and four

sloughed

I, but secondary

sloughed spermato-

or

GOSADOTROPINS

IN

MALE

375

LIZARDS

20+ 0-0

Spermatogonio

------o

Spermotocytes

A----A

Spermatids

I

IS-

,

, , ( /

1.0 Dose

( , 10.0

( pg I injection

50

I 100

400

i

12. Influence of various doses of ovine (NIH) FSH on the production and maturation of germ cells ir. caroli~nensis in September. Animals were injected every other day for 15 days (31°, 6 hr. of li except the highest dose which was given daily. Values represent the average mean number of cells in five cross sections of testicular tubules in five animals from each group. In uninjected controls there were five to ten spermatogonia per cross section and no other germ cells. FIG.

Anolis

,-.

,

Sex

Segmentof

I’ ,I’

x f

I

Epididymis

The combined dose of 10 pg FSH and LH was significantly less effective thani 10 ,JJ~ of FSH alone, since the former did not produce a significant development i~ any aspect of the sexual segment of the kidney. Thus, the antagonism of k FSH activity indicated by testis IS is aIs evident in these measurements of androgen production.

,05o!Jg FSH 170

Kidney

pr=

__--

-/--

.-

p

70 2 t 65

15 Time (days) FIG. 13. Development of the accessory sexua structures in physiologically hypophysectomized An&s treated with ovine (NIH) gonadotropins at 31”. The points indicate average mean values for six to eight animals. Doses (amount given every other day) are shown at the right. Uninjected controls showed no development during the 29-day period. Measurements are in micrometer units: 1 unit = 8 *.

z

Exp. 3: Testicular Recrudescence at f?O”

In 30 days the testes of normal lizards kept at 20” were equally enlarged by ali doses of either FSN (10 and 50 pg) or LH (50 and 100 pg), with an average weigbt of about 25 mg (control testes were at the initial size of 4 mg), Howev mortality had occurred in the groups. By 60 days the testes maining FSH-treated animals had increased to 35 mg, and there was s difference between the two doses ( 50 /Gil* The large increase In testis weights in these animals is partially explained by 91e edematous appearance of the gonads (Figs. 18-19). Despite the enlarged size, the o evidence of spermatogenic activity at

.--_^_FIG. 9. 4.0 fig FSH for 29 days; normal appearing early spermatids line the lumen. F’IG. 10. 10 pg FSH for 15 days; early spermatids are numerous around the lumen. FIG. Il. 10 pg FSH for 29 days; all stages of spermatogenesis including numerous spermatozoa are evident.

376

LIGHT

AND

PEARSON

FIGS. 14-17. Photomicrographs of accessory sexual structures in Anolis to show the effects of hormone treatment in physiologically hypophysectomized animals at 31’ (see quantitative values in Fig. 13). FIG. 14. Epididymis in lizard injected with 1 fig FSH on alternate days for 29 days. Appearance is the same as in untreated controls. FIG. 15. Epididymal tubules after 29 days of treatment with 10 ,ug FSH. The diameter and epithelial height of the tubules are greatly enlarged but no sperm are present in the lumina. FIG. 16. Cross section of the kidney in an animal treated with 1 pg FSH for 29 days. Appearance doesnot ,differ from controls. The vas deferens (vd) and sexual segment of the kidney (ssg) are indicated. FIG. 17. Cross section of the kidney in an animal treated with 50 fig FSH for 15 days. Only a few of the .enlarged sex segments (ssg) are indicated.

days was the production of a small number #of primary spermatocytes. The seminiferous tubules were greatly expanded but had very few germ cells at 30 days (Fig. 18). “rhere was some increase in the numbers of primary spermatocytes in both FSHtreated groups at 60 days, but spermatids were still sparse (Fig. 19). Both doses of FSH at 20” produced a significant (p < .02) enlargement of the epididymis and sexual segment of the kidney at 60 days whereas the high dose of LH (100 pg) produced about the same ef-

fect at 30 days. The extent of the development of the sex segment in these 20” groups was comparable to that in the 10 ,pg FSH groups at 31” after 30 days. Thus, there is clearly an androgenic response to gonadotropins at the lower temperature. In hypophysectomized lizards kept at 20”, 25 pg FSH produced an edematous testis as before. However, with an FSH dose of 1 pg every other day the increase in testis weight was identical to that of intact controls (Fig. 20)) and histologically the testes appeared normal.

GONADOTROPINS

FIGS. 18-19. Photomicrographs ca~ro3inenris kept at 20” starting

illustrating in September.

IN

MALE

LIZARDS

the effects of ovine Scales indicat,e 50 ,u.

377

NH!

FSH

on the

testes

in Ar&&

Fxo. 18. Testicular tubule is greatly expanded but, with only a few spermatogonia (sg) after 30 days t.reatmene with 10 fig FSH (compare scale with uninjected control in Fig, 6‘1. FIG. 19. Tubule is still enlarged and edematons in appesrsnce bllt there has been an increase in primary spermatocytes (XI) and some sperm&ids (sptd) after 60 days trestment, with 10 fig FSH at 20”.

DISCUSSION

50: i

25 days

Ot 20°C

1

FIG. 20. Effects of ovine (NH) FSH on testis weights in hypophyseetomized Anolis kept at 20”. Vertical bars indicate mean and thin vertical lines show the 95% confidence limits about the mean; each group consisted of seven to nine individuals. Injections were administered every other day. Without hormone treatments, the testis weights would be expected to be about 300/, of initial controls (Licht and Pearson, 1969) ~

Relative

Potencies

of PSH and LN

Mammalian FSH and LH are each capable of maintaining and stimulating both spermatogenesis and interstitial cell aetiviby in the lizard testis, although FSH is clearly the more potent. Spermiation was the only major aspect of testicular function t,hat we did not observe in our studies. It is likely that this failure was due to an insufficient period of treatment rather than t,o a lack of “spermiating” activit’y in the hor,mones t,ested. In studies with PMS in An&s (see discussion below; Licht,, 1967~) ) spermiation was not observed after 4 weeks but had occurred normally in all animals after 6 weeks. Quantitative comparisons of different, hormones is difficult without precise informat’ion on their purity. The difference between the potencies of FSN and EH, for

378

LICHT

AND

example, is probably much greater than the 20-fold estimate indicated by weight-comparisons of the two NIH preparations. The results obtained with the Papkoff-FSH indicate that the NIH-FSH standard contains only about 1% active FSH molecules, and Papkoff (personal communication) has indicated that an even more potent FSH preparation, with about 3 times the potency of the one used here, can be obtained, i.e. perhaps 300 X NIH standard. On the other hand, the most active LH preparations are only about 2,3 times that of the NIH-LH standard (Papkoff, 1966). Thus, ovine FSH may be many hundred times more potent, than ovine LH in promoting testicular activity in the lizard. It should be noted that the estimated potency of the Papkoff-FSH in the lizard (about 124 X NIH-FSH standard) is about &fold greater than its estimated relative potency based on the Steelman-Pohley assay (Table 1). In contrast, the neuraminidase treated Papkoff-LH had only about half the activity of the NIH-LH standard in the lizard, which is below its estimated LH activity in the rat (Table I). This reduction in activity may be due to a small FSH contamination since the lizard is highly sensitive to FSH and there is a possibility of synergism at low levels (Fig. 5). The problem of contaminants in the hormone preparations cannot be fully resolved at present. It seems likely that LH has some independent action in the male lizard since neuraminidase treatment should have removed all FSH activity. Papkoff (1966) has discussed the problem of determining the LH contamination in his FSH preparation. Recent tests (Papkoff, Personal communication) indicate that this contamination is less than 0.00017 unitsimg FSH; thus, it seems unlikely that LH contaminants could have been significant for the pronounced testicular responses to O.l-pg doses of this FSH. In addition to profound differences in gonadotropic potency, mammalian LH appears to have more deleterious side-effects than does FSH in the lizard. The basis for the high mortality associated with LH treatment is unclear but it was associated

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with stressful conditions. For example, it occurred in hypophysectomized animals (Exp. 1) and in intact animals kept at subnormal temperatures (Exp. 2). Comparison of testicular development rates in hormone-treated and intact animals allows some estimate of the hormone dose that is most comparable to physiological levels of gonadotropin. Such information should be useful for future studies of the gonadotropins. Testicular recrudescence in Anolis can be stimulated by long photoperiods and high temperatures (30-32’) during the fall (Licht, 1967a,b). The rate of testis growth under these conditions varies somewhat depending on the time at which treatment is begun, but in general, endogenous rates are comparable to those obtained with 5-10 fig (6-22 mu) NTHFSH given every other day at 31’. Normal testicular development at 20” is best duplicated by a lower dose, about 1 pg FSH. 0 ther Gonado tropins

Studies of the interaction between prolactin and gonadotropin in A. carolinensis (Licht, 1967c) provide additional information on gonadotropins in this lizard: daily injections of 25 pg ovine prolactin, 1 IU serum gonadotropin (PMS), or both were given under the same conditions and at, the same time of year as in Exp. 2. The effects of the prolactin on the testes were comparable to that observed with 0.1 pg (0.125 mu) NIH-FSH (Fig. 7). NIH bioassay data indicated a maximum FSH contamination of 0.025 mU in the prolactin dose used. Since the prolactin was given daily and since FSH may have a different potency in the lizard than indicated by mammalian bioassay (see discussion of Papkoff-FSH) it is not unlikely that the effect was due in part to prolactin contaminants. The effect of 1 IU PMS daily on testis weight and accessory structures was identical to that observed with 10 pg NIH-FSH (12.4 NIH mU or 0.33 IU) given every other day, and greater than that observed with 100 pg (98 NIH mU or 150 IV) NIH-LH. Thus, one unit of PMS is considerably more potent than an inter-

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national unit of LH but has less than a third the potency of one unit of FSH. The relative potencies and actions of porcine FSH and LH in the lizard Lacerto sicula reported by Della Corte and Cosenza (1965) differ in several respects from those observed in Anolis. However, their results are difficult to evaluate since they used relatively massive doses (5 mg every 3 days) of hormone preparations of undefined potency and purity, at relatively low and variable temperatures, and since endogenous gonadotropic activity was evident, in their int’act animals. dndroyens The role of androgens in vertebrate spermatogenesis is still unclear (see review by van Tienhoven, 1968). Gonadotropine may act through the stimulation of androgen production in interstitial cells with the -androgens exerting a more direct influence on the seminiferous epithelium. Testosterone treatment did not promote spermatogenesis in Anolis alt#hough it had a significant effect on the maintenance of the seminiferous epithelium following hypophysectomy (see Exp. 1). It has also been shown that similar testosterone treatment will not initiate recrudescence in the lizard Laceda sicula, and in fact, it inhibits this process (Licht et al., 1969). Testosterone and estradiol also caused a reduction in int8erstitial cell steroid dehydrogenase in Lacerta (Botte and Delrio, 1967). These effects may be due to a block of gonadotropin release at the hypothaIamic leve1 (L&k, 1967). However, the possibility exists t’hat the doses of testosterone were too low to promote spermatogenesis even though systemic levels were high enough to stimulate the accessory structures (see Sundaraj and Nayyar, 1967). Compnrisons with Other Vertebmtes The responsivenessof the male lizard to mammalian FSH and LH differs from that. observed in most other non-mammalian vertebrates (see reviews in Hoar, 1966; Knobil and Sandier, 1963; Nalbandov, 1966; Earner and Follett, 1966; van Tienhaven, 1968). In most fish the testes are

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considerably more sensitive to mammalian LB than to FSH and there is an increasing acceptance of the view that, the fish possess only a single LH-like gonadotropin. Male amphibia tend to show relativeIy discrete response to FSH and LN, the former stimulating spermatogenesis and the latter interstitial cell activity and especially spermiation (see also Lofts, 1961) ~In birds, both FSH and LH may affect various aspects of testicular activity but LH is generally more effective than FSH. The lizard studied here thus differs from all of these other groups in showing similar qualitative responses to both FSH and LH but being more sensitive to FSH in all respects, Hormonal Codrol of Season.d Reproductive Phenomena At high temperatures the growth of the testis and associated increases in the numbers of germ cells are highly dependent on the concentration of gonadotropin. In particular, spermatid formation may ho blocked at very low hormone doses, due to a differential mortality of sperm&ids or from a retarded rate of spermatocyte development. Sperm&ids also appeared to be the germ cells most sensitive to hypophysectomy in Anolis (Licht and Pearsoni 196’9). In many reptiles, the testes son-lain only spermatogonia and a few spermatocytes t,hroughout the faI1 and winter, even if temperatures are high (reviewed in Licht et al., 1969). This condition could result from relatively iow FSH levels. Developmental rat,es of accessory sexual structures (reflecting interst’itial celi activity) also depend on gonadotropin concentrations, and this process recuircx higher doses of hormone than are nieded to initiat,e spermatogenesis at 31”, Maintenance of the accessory strue,tures probably requires lesshormone than their initial development (cf. Exp. 1 and 2). In general, the relative dose re!ations observed among the different phases of spermatogenesisand androgen activity with exogenous ES are the same as occur when endogenous gonadotropin Ievels are modified by removal of different, amounts of the pars distalis (Licht, and Pearson, 1969).

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The results at 20” suggest that endogenous gonadotropin levels are normally relatively low at this temperature. Low temperatures may retard the rate of spermatogenesis and accessory sexual structure development at the target tissue level. It is clear that the testes are capable of responding in both respects, and that accessory structures can develop more rapidly than germ cells (Exp. 3). However, this development was only observed in response to hormone doses (lo-50 pg FSH) too high for normal spermatogenic development of the testes. The dose of FSH (1 pg) that best mimics the normal pattern of testis development at 20" is apparently too low to promote the development of accessory sexual structures. Licht (1967a) sugested that the lack of interstitial cell stimulation at 20” might be due to their insensitivity to gonadotropins at this low temperature. The present data suggest that the lack of interstitial cell activity at low temperatures may be due largely to a suppression of gonadotropin release. This conclusion is also supported by the fact that the sex segment of the kidney becomes atrophic while the testes continue to grow if intact animals are transferred to 20” in the spring after the testes and accessory structures are already fully developed (Licht and Pearson, 1969). It seems possible that a single gonadotropin (or gonadotropin complex) could testicular activity regulate seasonal through a combination of effects including an effect of temperature on gonadotropin release and a differential dose-sensitivity for spermatogenesis and androgen production. The gradual enlargement of the testes during the late fall and winter would result from low levels of gonadotropin which occur under the influence of low temperatures ; this condition would prevent the development of sexual accessory structures. Rising temperatures in the spring would stimulate gonadotropin release and these increased levels would lead to the rapid completion of spermiogenesis and a hypertrophy of the epididymis and sex segment of the kidney; the gonads would also respond rapidly due to their higher tem-

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perature. Gonadotropin levels might drop somewhat following this abrupt rise in the spring since the maintenance of the active gonadaI condition apparentIy requires Iess gonadotropin than does its initial development. This scheme contrasts with the one previously suggested for reptiles, namely, that fall recrudescence is regulated by FSH whereas a separate LH molecule is responsible for the stimulation of interstitial cell activity in the spring (e.g., Grignon and Grignon, 1965). The numerous difficulties involved in the determination of whether a single gonadotropin-complex or two separate gonadotropins exist in an animal are reviewed by van Tienhoven (1968). We cannot rule out the possibility that the lizard produces two separate hormones and that the activities of both are mimicked by the mammalian (ovine) FSH molecule. It is also important to emphasize that the present data concern only the male lizard, but preliminary evidence (Licht, unpublished) indicates similar gonadotropin responses in the female. The present data provide further support for the view (Licht and Pearson, 1969) that there may only be a single gonadotropin in the lizard. ACKNOWLEDGMENTS We thank Lynn Bengston for technical assistance and Emily Reid for the preparation of figures. We are particularly indebted to Dr. Harold Papkod for generously supplying gonadotropin prepara,tions and for his advice in the preparation of the manuscript. We are also grateful to The Endocrinology Study Section of the NIH for the hormones with which they provided us. The Ethicon corporation supplied the tissue adhesive (IBC-2) used in the surgery involved in hypophysectomy. This work was supported in part by NSF Grant GB-7366. REFERENCES C. I. (1967). “Statistics in Biology,” Vol. 1. McGraw-Hill, New York. BOTTE, V., AND DELRIO, G. (1967). The effect of estradiol-178 on the distribution of 3,&hydroxysteroid dehydrogenase in the testes of Rano esculenta and Lacerta sicula. Gen. Comp. Endocrinol. 9, 116-115. DELLA CORTE, F., AND COSENZA, L. (1965). Effetti de]I’LH e dell’FSH sui testicoli di Lacerfa S. BLISS,

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Sot. Pelor11, 113-122. DONINI, P., PUZZUOLI, I., LUNENFEL,D, B., ESHKOL, A., AND PARLOW, A. F. (1966). Purification and separation of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from human postmenopausal gonadotropin (HMG). Acta Endocrinol. 52, 169-185. FaRNnR, ]D. S., AND FOLLETT, B. K. (1966). Light and other environmental factors affecting avian reproduction J. A&m. 8ci. (Suppl.) 25, 96115. FOX, W. (1958). Sexual cycle of the male lizard, Anolis carolinensis. Cop&a 1958, 22-29. GRIGNOX, G., AND GRIGNON, M. (1965). Variations cycliques de l’activitd des glandes endocrines chez les rept.iles. Proc. 2nd Intern. Congr. Endocrinol. Part I., pp. 106-113. Ercerpta. Medica Int. Congr. Ser. No. 83. HOAR, W. S. (1966). Hormonal activities of the pars distalis in cyclostomes, fish and amphibia. In “The Pituitary Gland” (G. W. Harris and B. Donovan, eds.), Vol. I, pp. 242-294. University of California Press, Berkeley. %VOBIL, F., .~ND SANDL~R, R. (1963). The physiology of the adenohypophyseal hormones. In “Comparative Endocrinology” (U. S. von Euler and H. Heller, eds), Vol. I., pp. 447491. Academic Press, New York. LIGHT, P. (1967a). Environmental control of annual testicular cycles in the lizard Ano& carolinen.sis. I. Interaction of light and temperature in the initiation of testicular recrudescence. j. E~pptE. Zool. 165, 505-516. LIGHT, P, (1967b). Environmental control of annual testicular cycles in the lizard Anolti caroli~zensis. II, Seasonal variations in the effects of photoperiod and temperature on testicular recrudescence. J. ExptZ. Zool. 166, 243253. LIGHT, P. (1967~). Interaction of prolactin and ponadotropins on appetite, growth, and tail regeneration in the lizard, Anolis carolinensis. Gen. Comp. Endocrinol. 9, 49-63. LIGHT, P., HOYER, 33. E., .~ND VAN OQRDT, P. G. W. J. (1969). Influence of photoperiod and temperature on testicular recrudescence and body growth in the lizards, Lacerta siczda and Lacerta mura1is.J. Zool. 157, 469L501. LICIT, P., .~LND PEARSON, A. (1969). Effects of

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adenohypophysectomy on testicular function in the lizard Anolis carolinensis. Biol, Reprod. Hz, 107-119. LISR, R. D. (1967). Neural control of gonad size by hormone feedback in the desert iguana Dipsosauru dorsalis dorsalis. Gen. Comp. E&ocrinol. 8, 258-266. L~;OFTS, B. (1961). The effects of follicle-stimcl&ing hormone and luteinizing hormone on the testis of hypophysectomized frogs (Rana lemporaria), Gen. Comp. Endoerinol. 1, 179-189. N.~LBANDOV: A. V. (1966). Hormonal activity 5I’ the pars distalis in reptiles and birds. In ‘The Pituitary Gland” (6. W. Harris and B. Donovan, eds.), Vol. I, pp. 295-316. University of California Press, Berkeley. PAPKOFF, H. (1966). Recent studies on the purification and properties of ovine, bovine, and human interstitial cell stimulating hormone (ICSH, LH) and ovine follicle stimulating hormone (FSH). Proc. 6th Pun-Am. Congr. Endocrinol., iMexico City, pp. 334-339. Excerpta Medica Intern. Congr. Ser. No. 112. PA~KOFP, H., GOSPODAROWI~Z, D., CANDIOTTT, A., ?IND LI, 6=. H. (19%). Preparation of ovine intcrstitial cell-stimulating hormone in high yield. kch. Biochem. Biophys. 111, 431438. PAPKOFF, H., GOSPOMROWICZ, D., ANXS Lr. C. 13. (1967). Purification and properties of ovine follicle-stimulating hormone. Arch. Biochem. Biophys. 120, 434439. PRASAD,

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Steroids in relation to reproduction in lizards. Proc. 2nd Intern. Congr. Hormonal Sts~oids~ pp. 1065-1072. Excerpta Medica Int. Gong. Ser. No. 132. SAINT GIRONS, H. (1967). Morphologie comparee de l’hypophyse chez les squamata. Bnn. Sd. Nat. Zool. 9, 229-308. SUNDARAJ, B. I., AND NAYYAR, S. K. (1967). E?t’ects of exogenous gonadotrophins and gonadal hormones on the testes and seminal vesicles of hypophysectomized catfish, Heteropneustes fossiZ.s (Block). Gen. Cow&p. EndoerznoE. 416. vm TIENHD~N, A. (1968). “Reproductive physiology of Vertebrates.” Saunders, Philadelphia, Pennsylvania.