0013-7227/90/1272-0747$02.00/0 Endocrinology Copyright ca 1990 by The Endocrine Society

Vol. 127, No. 2 Printed in U.S.A.

Regulation of Insulin-Like Growth Factor I and Stage-Specific Levels of Epidermal Growth Factor in Stage Synchronized Rat Testes* J. M. S. BARTLETT, J. SPITERI-GRECH, AND E. NIESCHLAG Max Planck Clinical Research Unit for Reproductive Medicine and Institute of Reproductive Medicine of the University, D-4400 Munster, Federal Republic of Germany

ABSTRACT. Stage synchronization of seminiferous epithelium after withdrawal and replenishment of vitamin A provides a valuable and powerful approach to the investigation of paracrine interactions within the testis. However, since the discovery of this model, little attention has been given to the events surrounding the synchronous reinitiation of spermatogenesis after depletion of vitamin A. Synchronization of spermatogenesis was observed in all animals previously deficient in vitamin A. However, the degree of synchrony observed, as assessed by a ratio of synchrony, decreased markedly with time. The possibility that spermatogenic synchrony decreases with time due to variability of the temporal duration of stages of the cycle of the seminiferous epithelium is supported by this observation. However, long-term studies are required to substantiate this point. After initiation of stage synchrony of spermatogenesis, increased testicular concentrations of epidermal growth factor (EGF) were observed in testes synchronized between stages IX-

II than at other stages of the cycle of the seminiferous epithelium. This elevation in testicular EGF concentrations correlated well with mitotic division of type A spermatogonia at stages IX, XII, and XIV of the cycle of the seminiferous epithelium. Previous in vitro studies have implicated an EGF-like factor in the stimulation of type A spermatogonial division in the mouse. A significant increase in testicular insulin-like growth factor I (IGF-I) concentrations was observed in control animals 14 days after the injection of retinol acetate. In vitamin A deficient animals, a marked increase in testicular IGF-I concentrations was observed as compared to age-matched controls. Maximal levels of testicular IGF-I concentrations were present 14 and 28 days and again 126 days after re-supplementation with retinol acetate. No stage dependent changes in testicular IGF-I were observed but the data provided suggest the retinol may be one of the factors involved in the regulation of testicular IGF-I. (Endocrinology 127: 747-758,1990)

O

VER recent years, interest in the paracrine regulation of testicular function has grown rapidly. The study of Sertoli-germ cell interactions, in particular, could result in advances in our understanding of testicular function and dysfunction (1). In vitro studies on spermatogonial development have demonstrated that vitamin A and FSH regulate the production of a peptide that stimulates spermatogonial stem cell division (2, 3). Further studies have suggested that this factor may be related to EGF (4). EGF receptors have been demonstrated on many testicular cell types (5) including Sertoli and Leydig cells. In addition, EGF has been shown to stimulate Sertoli cell lactate production in vitro (6). The possibility exists that EGF or an EGF-like peptide may be involved in the initiation of synchronous spermatogenic development as well as in normal testicular function. The presence of insulin-like growth factor I (IGF-I) in

testicular extracts and Sertoli cell culture medium has been established by many groups (7-10). Messenger RNA (mRNA) for IGF-I has been also seen isolated within the testis (11, 12). FSH, LH, and GH have all been shown to modulate IGF-I mRNA in immature female hypophysectomized rats (13). Receptors for IGF-I have been isolated within the testis (8, 14). IGF-I has been shown to be present in or secreted from Sertoli cells and primary spermatocytes (15, 16). Production of IGF-I by Sertoli cells is regulated by FSH, GH, and growth factors (10, 14, 17). IGF-I actions on Sertoli cells have been shown to include thymidine uptake, glucose uptake, and lactate production (18-20). In addition, IGF-I has been postulated as a general regulator of FSH action (21). The use of vitamin A-induced synchronization of the cycle of the seminiferous epithelium to study paracrine testicular events has increased since initial reports of this phenomena were published (22-24). In the present study, we investigated the role of EGF and IGF-I in the regulation of germ cell development within the testis. The effects of vitamin A withdrawal and subsequent replacement on EGF and IGF-I concentrations and on

Received January 2, 1990. Address reprint requests to Professor E. Nieschlag, Institute of Reproductive Medicine of the University, Steinfurter Strafte 107, D4400 Munster, Federal Republic of Germany. 747

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GROWTH FACTORS IN SYNCHRONIZED SPERMATOGENESIS

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the qualitative histology of the rat testis over a period of more than 4 months are reported.

Materials and Methods Animals Thirty-six pregnant female Sprague-Dawley rats (Zuchtinstitut, Hannover, West Germany) were housed singly and separated into two groups. Eighteen animals whose pups were to be maintained on a vitamin A-free diet were allowed free access to a casein-based pelleted vitamin A-free diet from day 20 of gestation (Sniff, Soest, West Germany). Eighteen control females and pups were allowed free access to the same diet supplemented with 0.0333 mg retinol acetate/g (to give an approximate daily dose of 0.5 mg retinol acetate/rat/day based on an average food consumption of 20 g/rat-day). The diet was prepared as described previously (24). No effect of treating pregnant females on liter size (10.7 ± 0.5 us. 10.0 ± 0.7, NS, P > 0.05, control us. treated), sex ratio (1.14 us. 1.14 male-female) or numbers of animals giving birth (15/18 us. 15/18, control us. treated) were observed as a result of this treatment. Treated animals were maintained on a vitamin A-free diet for 9 weeks, when all animals were injected with two sc injections of retinolacetate suspended in glycerol as previously described (24). Subsequently, all rats received vitamin A-free diet supplemented with retinol acetate. In order to assess the long-term effects of vitamin A deficiency and restoration on stage synchronization, and to determine whether such synchronization was stable, animals were killed immediately before injection of vitamin A and 14, 28, 42, 56, 70, 84, 98, 115, 119, 122, and 126 days after resupplementation of vitamin A. Control and treated animals were anaesthetized with CO2 and decapitated. Trunk blood and pituitary, testis, and epididymal tissues were collected for hormonal and histological evaluation. Histological evaluation was performed on six to seven treated animals per group and six to seven control animals randomly selected from the respective groups. Testosterone assay Serum and testicular testosterone concentrations were measured by luminescence-immunoassay (LIA) after ether extraction as described previously (25, 26). The mean intraassay variations were 5.2% and 6.7% and the interassay variations were 8.1% and 9.4% in the ranges 10-12 and 19-21 nmol/liter, respectively.

Endo • 1990 Vol 127 • No 2

RIA for EGF

A commercial RIA for human EGF (Amersham, Braunschweig, West Germany) was validated for the measurement of rat testicular EGF. A purified preparation of rat EGF (Bioproducts for Science Incorporation, Indianapolis, IN) was obtained and dilution curves for normal rat submandibular gland and testicular EGF shown to be parallel to the standard curve. The limit of detection for the assay was 1.6 ng/ml, with an intraassay coefficient of variation of 5.6% and an interassay coefficient of variation of 10.2%. RIAofIGF-I A commercial RIA for human IGF-I (Amersham) was validated for the measurement of rat serum and testicular IGF-I. A standard curve for human IGF-I in the range 0.5-250 ng/ml was established using recombinant human IGF-I (Amersham). Normal rat serum and testicular homogenates were acidethanol extracted (27), and dilution curves were shown to be parallel to the standard curve. Results are expressed in terms of nanograms of pure human IGF-I/ml assuming equal crossreactivity of rat and human IGF-I in the RIA. The limit of detection for the assay was 2.5 ng/ml, with an intraassay coefficient of variation of 2.9% and an interassay coefficient of variation of 12.9%. Testicular histology Testes were fixed in Bouin's solution, dehydrated, and embedded in Paraplast (Lancer, Athy, Ireland) and stained with periodic acid-Schiff s reagent and hematoxylin. Two hundred seminiferous tubules per animal were classified according to Leblond and Clermont (28). Tubules devoid of elongated spermatids were identified by the morphology of the acrosome of round spermatids or by spermatogonia and spermatocyte populations where present. Percentages of tubules at any one stage were estimated after subtraction of the total number of damaged tubules (those tubules too regressed to be classified according to stages were described as "damaged"). Numbers of damaged tubules are shown as a percentage of the total number (n = 200) of tubules counted per animal. A ratio of synchrony was calculated as follows: The stages over which synchrony was observed were classified as those stages showing at least 10% of all classified tubules, or inclusive of stages lying between two such stages. The percentage sum of tubules represented within synchronized stages under this classification was then divided by the percentage sum of tubules present within such stages in control animals to produce a ratio reflecting the degree of synchrony achieved.

Pituitary hormone assays Serum and pituitary LH, FSH, PRL, and GH were measured using RIA with reagents supplied by the National Institute of Arthritis, Kidney and Digestive Diseases (Bethesda, MD). The standard preparations used were FSH-RP-2, LH-RP-1, rat (r)GH-RP-2 and rPRL-RP-3; tracers were prepared from rLH1-6, rFSH-I-6, rGH-I-5, and rPRL-I-5 and the antisera were anti-rLH-S-9, anti-rFSH-S-11, anti-rGH-S-5, and anti-rPRLS-9. The limit of detection in all assays was 0.16 ng/tube. The mean intraassay variations were 7.5%, 7.3%, 7.3%, and 6.3%, and the interassay variations were 9.5%, 6.7%, 9.8%, and 2.6% for FSH, LH, GH, and PRL, respectively.

Statistics Data were analyzed using analysis of variance ANOVA and Students t tests to determine significant differences. The level of significance was set at 5%. Data are expressed as mean ± SE.

Results Growth and food consumption Both control animals and animals treated with vitamin A-free diet showed identical growth up to 6 weeks of age,

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GROWTH FACTORS IN SYNCHRONIZED SPERMATOGENESIS at which point, growth in vitamin A-deficient animals slowed appreciably between weeks 6-9. Between weeks 8-9, control animals showed an average increase in body wt of 38.5 g/rat whereas treated animals showed an average increase in body wt of only 7.8 g/rat. From 6-9 weeks of age, weight gain was 31.7 g/rat in treated animals as compared to 146.4 g/rat in control animals. At 9 weeks of age (day 0 for experimental purposes), retinol acetate was injected into both treated and control animals and between 9-11 weeks of age (day 0-14), treated animals showed an increase in body wt of 86.5 g/ rat as compared with an increase of 48.2 g/rat in control animals. Thereafter, between 11-21 weeks of age (day 14-84), growth in both groups proceeded at similar rates, although animals that were previously vitamin A deficient weighed consistently less than control animals. From 22 weeks of age to the termination of the experiment at 25 weeks of age (day 84-126), growth in animals previously deficient in vitamin A ceased, while control animals maintained a steady wt increase. At termination of the experiment, treated animals weighed on average 158 g/rat less than untreated control animals. Throughout this period, food consumption by treated animals was marginally lower than that observed in controls. A reduction in food consumption in line with the reduction observed in body wts may suggest that food consumption/body wt was relatively constant in these animals (data not shown). Organ wts Pituitary wts were significantly lower (P < 0.05) in vitamin A-deficient animals immediately before and up to 14 days after vitamin A restoration (Table 1). Similarly, at 98 days and then from 119-126 days post vitamin A replenishment, pituitary wts in treated animals were significantly lower than those observed in controls (Table 1). In both control and treated animals, pituitary wts increased significantly with time (Table 1). At all times after vitamin A depletion and restoration, testicular wts were significantly (P < 0.05) reduced after vitamin A depletion to less than 50% of control values, with a decrease to below 40% of control values 14 days after vitamin A restoration. A significant (P < 0.05) increase in testicular wts in treated animals above values from vitamin A-deficient animals (day 0) was observed between 56 and 119 days post vitamin A restoration. Thereafter testicular wts fell to levels not significantly different from values in vitamin A-deficient animals (Table 1). Paired epididymal wts were significantly (P < 0.05) reduced after vitamin A depletion by approximately 2030%. No increase in epididymal wts was observed in treated animals post vitamin A replenishment, while in

749

TABLE 1. Body and tissue wts Time (days) Day 0 Control Treated Day 14 Control Treated Day 28 Control Treated Day 42 Control Treated Day 56 Control Treated Day 70 Control Treated Day 84 Control Treated Day 98 Control Treated Day 115 Control Treated Day 119 Control Treated Day 122 Control Treated Day 126 Control Treated

Body

Pituitary

Testicular

wt (g)

wt

wt

wt

(mg)

(mg)

(mg)

10.1 ± 0.7 7.1 ± 0.5°

2693 ± 29 1291 ± 191"

707 ± 29 561 ± 93°

6 6

398 ± 5* 10.9 ± 0.4 323 ± 16"'c 9.9 ± 0.6"

2913 ± 77 1115 ± 78"

1026 ± 46* 708 ± 44°

6 6

417 ± 35* 11.2 ± 1.0 353 ± 23a'c 10.3 ± 1.1

2988 ± 155 1298 ± 88°

1049 ± 83* 730 ± 38"

6 6

471 ± 25* 12.6 ± 0.5 415±28 a ' c 11.6 ± 1.0e

3518 ± 217" 1387 ± 88°

1201 ± 256 786 ± 33°

6 6

482 ± 14* 12.2 ± 0.3 385 ± 34"'c 11.2 ± l.l c

3532 ± 200* 1432 ± 68" 2001 ± 223°'c 983 ± 70"

6 6

523 ± 19* 11.7 ± 0.7 422±48 a ' c 10.6 ± 1.2

3166 ± 161 1372 ± 19* 7 2123 ± 249°'c 1012 ± 128" 6

551 ± 276 11.9 ± 0.9 443 ± 20°'c 11.0 ± 0.8c

1602 ± 60* 3430 ± 87* 1990 ± 289OC 1061 ± 79°

325 ± 9 213 ± 30°

Epididymal n

7 6

561 ± 18* 13.1 ± 0.5" 3371 ± 70* 1576 ± 42* 7 473 ± ll a ' c 12.0 ± 0.9OC 2352±433°' c 1238 ± 185° 6 526 ± 27* 13.2 ± 0.9* 427 ± 56a'c 12.4 ± 1.9"

1571 ± 100* 7 3467 ± 90" 2192±249°' c 1227 ± 191" 6

1547 ± 72* 7 553 ± 26* 12.5 ± 0.9 3335 ± 87" 446±34 a c 11.0 ± 0.8°'c 2277 ± 260°'c 1237 ± 103° 7 587 ± 18" 14.5 ± 1.0* 3739 ± 211* 359±43 a c 10.8 ± l.l"' c 1622 ± 179"

1737 ± 103* 7 831 ± 105" 7

603 ± 29" 12.8 ± 0.9 410 ± 68°'c 11.5 ± 1.2C

1635 ± 142* 6 973 ± 162° 6

3379 ± 214" 1886 ± 311"

Values are given as mean ± SE. Time intervals represent time in days post injection of untreated (control) or vitamin A-deficient (treated) rats. a Significantly different (P < 0.05) from values obtained from agematched control animals. * Significantly different (P < 0.05) from values obtained from control animals at Day 0, i.e. before vitamin A injection (refers only to comparisons between control animals). c Significantly different (P < 0.05) from values obtained from vitamin A-deficient animals {i.e. treated animals at day 0 before injection of vitamin A, refers only to comparisons between treated animals).

control animals epididymal wts increased significantly with time. Pituitary gonadotropin concentrations Pituitary LH concentrations were significantly (P < 0.05) raised with respect to control values up to 70 days post vitamin A replenishment and again between days 122-126 post vitamin A replacement (Table 2). Pituitary FSH concentrations followed a similar pattern to that

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GROWTH FACTORS IN SYNCHRONIZED SPERMATOGENESIS

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Endo • 1990 Voll27«No2

TABLE 2. Pituitary and serum gonadotropins Serum

Pituitary Time Days DayO Control Treated Day 14 Control Treated Day 28 Control Treated Day 42 Control Treated Day 56 Control Treated Day 70 Control Treated Day 84 Control Treated Day 98 Control Treated Day 115 Control Treated Day 119 Control Treated Day 122 Control Treated Day 126 Control Treated

LH

FSH

PRL

GH

LH

FSH

PRL

GH

(ng/mg)

(ng/mg)

(ng/mg)

(ng/mg)

(ng/ml)

(ng/ml)

(ng/ml)

(ng/ml)

4291 ± 407 6166 ± 741°

552 ± 71 835 ± 115"

73.3 ± 9.0 77.7 ± 16.0

1347 ± 692 3322 ± 944

2.9 ± 1.6 3.8 ± 0.7

6.0 ± 0.8 9.5 ± 1.3

9.1 ± 2.5 4.6 ± 1.0°

18.5 ± 5.0 12.1 ± 4.9

4176 ± 431 5692 ± 679°

489 ± 29 563 ± 100

276 ± 50* 149 ± 13.9°

4.1 ± 1.1 7.0 ± 1.7°

7.5 ± 0.8 12.7 ±3.1

4.7 ± 1.2 3.4 ± 0.5

21.4 ± 2.9 25.5 ± 3.3

3572 ± 493 53 ± 809°

378 ± 50 518 ± 105°

182 ± 33 153 ± 23

3141 ± 1097 1292 ± 403

3.3 ± 1.2 7.9 ± 1.9"

6.2 ± 0.8 11.5 ± 1.9 °

6.5 ± 1.2 12.5 ± 4.3°

15.5 ± 4.4 16.9 ± 1.5

4081 ± 750 5063 ± 417°

343 ± 76 442 ± 65°'c

255 ± 35* 157 ± 23°

2973 ± 612 2493 ± 304

3.8 ± 1.1 13.2±1.3"'c

15.1 ± 8.9 19.4 ± 7.3

7.1 ± 1.6 8.4 ± 1.2

48.6 ± 22.6 20.5 ± 3.0

3115 ± 742 8122 ± 4467°

418 ± 64 474 ± 75

214 ± 29* 257 ± 31

10056 ± 8975 12324 ± 11724

9.2 ± 3.3* 6.8 ± 3.1

8.8 ± 1.6 8.8 ± 1.4

11.8 ± 1.9 7.6 ± 2.7°

22.2 ± 7.6 37.6 ± 16.4

2627 ± 551* 4451 ± 656 ac

240 ± 29* 383 ± 42°'c

252 ± 39* 181 ± 16°

1925 ± 394 2994 ± 1019

2.8 ± 0.4 5.1 ± 2.3°

4.8 ± 1.0 6.5 ± 1.4

7.6 ± 2.0 7.7 ± 1.8

13.1 ± 2.2 13.2 ± 3.3

5421 ± 842 4199 ± 500°

449 ± 54 378 ± 53°'c

367 ± 53* 148 ± 19°

4026 ± 1337 3238 ± 415

5.4 ± 2.2 4.4 ± 0.6

11.0 ± 4.2 9.0 ± 1.3

17.4 ± 2.6 10.0 ± 2.4°

24.8 ± 6.4 14.5 ± 1.8

2888 ± 493 3868 ± 453a

268 ± 30* 299 ± 45C

154 ± 34 148 ± 19

2688 ± 528 1848 ± 223

5.9 ± 1.5 8.9 ± 1.8°

5.9 ± 0.4* 10.8 ± 1.8

15.4 ± 3.4 16.0 ± 2.7C

24.4 ± 7.8 22.4 ± 5.2

2595 ± 313 2313 ± 622C

288 ± 27* 239 ± 38°-c

227 ± 84* 102 ± 41°

2547 ± 365 2260 ± 170

10.2 ± 2.1* 7.8 ± 2.2

9.4 ± 1.3 6.9 ± 1.2

20.0 ± 4.4 13.7 ± 2.6°

83.0 ± 61.7 168.9 ± 88.6

2845 ± 464 2645 ± 268C

285 ± 43* 280 ± 30c

170 ± 55 221 ± 61

3089 ± 685 2663 ± 155

6.8 ± 0.9* 6.6 ± 1.2

6.6 ± 0.5 7.1 ± 0.8

15.7 ± 3.1 7.5 ± 2.0°

18.3 ± 4.0 32.7 ± 9.2

1665 ± 208* 2786±240°-c

214 ± 17* 329 ± 34°'c

185 ± 52 175 ± 28C

2875 ± 516 1625 ± 243

8.7 ± 1.4* 10.4 ± 0.9

6.6 ± 0.7 13.1 ± 2.0

13.5 ± 3.4 9.2 ± 1.5°

41.0 ± 9.4 36.1 ± 7.6

1039 ± 142* 2378 ± 380OiC

270 ±41* 342 ± 83C

151 ± 45 113 ± 12

1593 ± 143 1937 ± 319

12.4 ± 2.2* 9.7 ± 2.4

6.2 ± 0.6 10.6 ± 1.4

45.6 ± 12.6* 20.2 ± 8.2°'c

43.6 ± 16.1 44.5 ± 10.5

1358 ± 591 795 ± 442

Data are obtained by RIA of homogenized pituitary tissue or untreated serum. Values are given as mean ± SE. Time intervals represent time in days post injection of untreated (control) or vitamin A-deficient (treated) rats. 0 Significantly different (P < 0.05) from values obtained from age matched control animals. * Significantly different (P < 0.05) from values obtained from control animals at day 0, i.e. before vitamin A injection (refers only to comparisons between control animals. c Significantly different (P < 0.05) from values obtained from vitamin A-deficient animals {i.e. treated animals at day 0 before injection of vitamin A, refers only to comparisons between treated animals).

observed for LH (Table 2), although significant increases in pituitary FSH concentrations were not as consistent as those observed for LH. Pituitary concentrations of both FSH and LH in both controls and treated animals fell significantly toward the end of the observation period. Pituitary concentrations of PRL were either similar to or significantly lower than control values. However, no consistent pattern of pituitary PRL changes could be recognized (Table 2). Pituitary GH concentrations showed no significant changes between controls and treated animals or throughout the time course of the current study.

Serum hormones Serum LH concentrations were not significantly elevated in vitamin A-deficient animals. However, up to 42 days after vitamin A replenishment, serum LH concentrations were significantly (P < 0.05) higher than control values (Table 2). Serum FSH concentrations were elevated before and up to 28 days post vitamin A replenishment, then remained at near normal concentrations up to 119 days post vitamin A treatment, rising again between 119-126 days post treatment (Table 2). Serum PRL concentrations were reduced in vitamin A-deficient

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GROWTH FACTORS IN SYNCHRONIZED SPERMATOGENESIS

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TABLE 3. Serum growth factors and testosterone animals and at a number of time points during the observation period (Table 2). Serum GH concentrations Serum T'irtto showed no significant changes between controls and i lme T IGF-I treated animals or throughout the time course of the (days) (nmol/liter) (ng/ml) current study. DayO No significant changes in serum testosterone values Control 13.9 ± 4.7 78.7 ± 3.5 were observed between controls and treated animals 62.4 ± 6.2" 6.2 ± 0.8 Treated during the study with the exception that, 122 days post Day 14 vitamin A treatment, serum testosterone concentrations Control 80.6 ± 3.8 15.0 ± 4.7 Treated 14.5 ± 5.5 82.9 ± 2.9 were lower in treated than in control animals (P < 0.05). Day 28 However, multifactorial analysis of variance of the data Control 13.0 ± 5.2 65.3 ± 5.0 revealed an overall negative effect of treatment (P < Treated 75.9 ± 3.4 18.8 ± 3.3 0.05) on serum testosterone values (Table 3). Day 42

Testicular testosterone Testicular testosterone concentrations in vitamin Adeficient animals were not significantly different from those observed in control animals. However, between 1442 days post vitamin A injection, testicular testosterone concentrations were markedly increased with respect to control values (P < 0.05, Table 3). Thereafter, no significant differences in testicular testosterone concentrations were observed between control and treated animals. EGF Testicular concentrations of EGF were significantly (P < 0.05) raised in vitamin A-deficient animals and for up to 42 days after vitamin A replenishment (Fig. 1). Between 56-119 days post vitamin A replenishment, testicular EGF concentrations were not significantly elevated in treated animals with respect to control animals. However, between 119-126 days post vitamin A replenishment, testicular EGF levels were once again significantly elevated with respect to control values (Fig. 1; P < 0.05). No correlation between numbers of damaged tubules and testicular EGF concentrations was observed. IGF-I Serum concentrations of IGF-I were not significantly elevated in treated animals either before or after treatment with retinol in vitamin A-deficient animals. A significant reduction in serum IGF-I was observed before vitamin A replacement and 98 days post vitamin A replacement. Serum IGF-I concentrations were significantly elevated in treated animals 6 weeks after vitamin A replacement (P < 0.05, Table 3). No correlation between serum and testicular IGF-I concentrations was observed, nor was any correlation between testicular IGF-I concentrations and the proportions of damaged tubules observed. Testicular concentrations of IGF-I were significantly (P < 0.05) elevated with respect to controls in vitamin A-deficient animals and at all time points after vitamin

Control Treated Day 56 Control Treated Day 70 Control Treated Day 84 Control Treated Day 98 Control Treated Day 115 Control Treated Day 119 Control Treated Day 122 Control Treated Day 126 Control Treated

Tocfiriilor T1

i esLicuidr I

(ng/g) 68.8 ± 14.6 8.3 ± 13.2 150.9 ± 23.8 274.3 ±46.7°'fc 114.2 ± 42.6 623.0 ± 122.0"'*

22.8 ± 4.2 12.3 ± 7.5

66.9 ± 1.9 79.8 ± 5.9°

207.0 ± 43.6C 519.0 ± 134.9"'"

15.0 ± 2.5 12.9 ± 7.1

73.5 ± 3.6 82.2 ± 3.4

143.0 ± 21.4 231.0 ± 105.2

14.9 ± 2.7 10.5 ± 3.5

67.7 ± 3.0 73.8 ± 3.8

134.9 ± 26.4 208.0 ± 77.7

15.2 ± 4.7 6.2 ± 1.0

67.5 ± 5.8 58.3 ± 3.4

122.2 ± 33.2 89.5 ± 41.8

9.0 ± 1.6 8.0 ± 1.8

66.3 ± 2.2 56.6 ± 2.8

10.8 ± 2.2 9.7 ± 1.8

64.0 ± 5.8 66.4 ± 8.9

83.2 ± 17.2 118.6 ± 21.3

a

84.6 ± 20.8 152.2 ± 45.5

6.2 ± 1.0

55.3 ± 2.1

48.4 ± 7.4

4.0 ± 1.1

57.5 ± 2.5

55.6 ± 16.0

7.5 ± 2.2 2.3 ± 0.6°

53.1 ± 4.0 56.6 ± 6.4

50.8 ± 17.8 54.7 ± 17.8

9.3 ± 2.5 5.9 ± 2.3

53.9 ± 3.0 63.1 ± 5.2

65.2 ± 19.8 102.7 ± 40.1

Data obtained by RIA of homogenized testicular tissue or untreated serum (testosterone and EGF) and by RIA of acid ethanol-extracted serum (IGF-I). Values are mean ± SE. Time intervals represent time in days post injection of untreated (control) or vitamin A-deficient (treated) rats. " Significantly different (P < 0.05) from values obtained from agematched control animals. 6 Significantly different {P < 0.05) from values obtained from vitamin A-deficient animals (i.e. treated animals at day 0 before injection of vitamin A, refers only to comparisons between treated animals). c Significantly different (P < 0.05) from values obtained from control animals at day 0, i.e. before vitamin A injection (refers only to comparisons between control animals).

A replacement (Fig. 1). In addition, a marked stimulation in testicular IGF-I concentrations was observed in treated animals 14 and 28 days post vitamin A treatment (P < 0.05) and also in control animals 14 days post vitamin A treatment (Fig. 1). Thereafter, vitamin A levels remained elevated above baseline levels in control animals throughout the observation period while values

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GROWTH FACTORS IN SYNCHRONIZED SPERMATOGENESIS

752

Endo« 1990 Voll27« No 2

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14 2B 42 56 70 84 98 115 119 122 126 Days post vitamin A injection

FlG. 1. Testicular EGF and IGF-I. Testicular concentrations (ng/g) of IGF-I {toppanel) and EGF (bottompanel), in vitamin A-treated control (solid bars) and vitamin A-deficient (open bars) rats. Values are given as mean ± SE. Time intervals represent time in days post injection of untreated (control) or vitamin A-deficient (treated) rats. *, Significantly different (P < 0.05) from values obtained from age-matched control animals, a, Significantly different (P < 0.05) from values obtained from control animals at day 0, i.e. before vitamin A injection (refers only to comparison between control animals), b, Significantly different (P < 0.05) from values obtained from vitamin A-deficient animals (i.e. treated animals at day 0 before injection of vitamin A) refers only to comparisons between treated animals).

in treated animals declined briefly before rising again toward the end of the observation period (Fig. 1). Stage synchrony and growth factor concentrations To analyze stage-specific changes in testicular growth factor concentrations, animals from 56-126 days post vitamin A replenishment (where full qualitative spermatogenesis was observed) were grouped according to stage distribution rather than age. Testicular growth factor concentrations from animals whose spermatogenesis was synchronized between stages; V-VII (n = 10), VII-VIII (n = 5), V-VIII (n = 4), IX-XIV (n = 5), XIIII (n = 8), XIV-II (n = 3), I-VIII (n = 5), and VI-XIV (n = 2) were calculated and compared (Fig. 2). In addition, growth factor concentrations in 18 animals syn-

N 5 IVIII

8

10 VVII

VII VIII

18

V-

K - XII XIV

VIII

XIV

-II

-II

VIXIV

DC—II

Stage

FIG. 2. Relationship between testicular EGF and IGF and stage synchronization. Testicular concentrations (ng/g) of IGF-I (top panel) and EGF (bottom panel), in animals displaying stage synchronization. N, Number of animals whose testicular morphology was synchronized at the stages denoted on the X-axis. Stage indicates stages of the cycle of the spermatogenic epithelium where synchronization was observed, i.e. those stages representing >10% stages observed or lying between two such stages. In animals with qualitatively normal spermatogenesis (open symbols) and those with incomplete spermatogenesis (closed symbols). *, Significantly different (P < 0.05) from animals with spermatogenesis synchronized between stages V-VIII of the cycle of the seminiferous epithelium. **, Significantly different (P < 0.05) from animals with spermatogenesis synchronized between stages IX-XIV of the cycle of the seminiferous epithelium, with qualitative normal spermatogenesis.

chronized between stages IX-XIV but in which spermatogenesis was not qualitatively complete (i.e. tubules containing germ cells up to the level of preleptotene spermatocytes or round spermatids only) were included in this analysis. Testicular concentrations of EGF were significantly higher (P< 0.05) in animals synchronized between stages IX-II of the cycle of the seminiferous epithelium (n = 16) than in animals synchronized between stages V-VIII (n = 19; 16.0 ± 1.0 us. 12.2 ± 0.8 ng/g, respectively) of the cycle of the spermatogenic epithelium (Fig. 2). In animals in which spermatogenesis was not qualitatively

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GROWTH FACTORS IN SYNCHRONIZED SPERMATOGENESIS complete (i.e. between 14-42 days post vitamin A replacement), spermatogenesis was assessed by the presence of preleptotene spermatocytes and at 42 days of round spermatids and was synchronized between stages IX-XIV in 16 animals and between IX-II in 2 animals. Mean testicular EGF concentrations in these animals (20.83 ± 1.12 ng/g) were significantly higher than in animals synchronized at stages IX-XIV with full spermatogenesis (P < 0.05) and than in animals synchronized around stages V-VIII of the cycle of the seminiferous epithelium (P < 0.05). In addition, mean testicular EGF concentrations in animals before vitamin A treatment were significantly lower (P < 0.05) than in comparative animals with incomplete spermatogenesis and spermatogenesis synchronized between stages IX-XIV. No overall significant stage-dependent changes could be observed in testicular IGF-I concentrations although concentrations in animals whose spermatogenesis was synchronized around stages VII-VIII were marginally higher than observed in other groups. Animals in which spermatogenesis was not qualitatively complete showed markedly higher testicular IGF-I concentrations than those in which spermatogenesis was qualitatively normal. Histology

Qualitative analysis of 78 of 81 control animals showed normal spermatogenesis and a normal distribution of stages of the cycle of the seminiferous epithelium. Three animals showed significant (30-80%) proportions of damaged tubules and were excluded from analysis for this reason. Twenty-four control animals were selected at random (2 from each time point), and stages of the cycle of the seminiferous epithelium were catagorized in these animals. Testicular sections after induction of vitamin A deficiency showed the presence of Sertoli cell and type A spermatogonia in all sections. In many tubule crosssections some preleptotene spermatocytes or type B spermatogonia could be observed and in two of the six animals in this group some evidence of degeneration, i.e. retention of spermatids or degenerating cells could be observed. Classification of stages within this group was largely not possible due to the severe degree of regression observed. Fourteen days after vitamin A replenishment, the majority of seminiferous tubules showed the presence of preleptotene spermatocytes in all animals. In three animals significant numbers of pachytene spermatocytes were observed. Classification of stages, based on the presence of preleptotene spermatocytes, suggested that the majority of tubules (99%) in all animals were synchronized between stages IX-XIV of the cycle of the spermatogenic epithelium.

753

Twenty-eight days post vitamin A injection, the majority of seminiferous tubule sections showed the presence of preleptotene spermatocytes and pachytene spermatocytes, with small numbers of tubules displaying the presence of elongating spermatids. Once again the majority of seminiferous tubules could be classified between stages IX and XIV of the cycle of the seminiferous epithelium. Forty-two days after injection of vitamin A, the majority of seminiferous tubules contained spermatids at early stages of development. Four animals showed strong synchrony between stages IX-XIV of the cycle, a single animal showed synchrony to a lesser degree between stages IX-I of the cycle while the final animal within this group displayed a lesser degree of synchrony between stages IX-II of the cycle of the spermatogenic epithelium. Fifty-six days after replacement of vitamin A, the extent of recovery of spermatogenesis was such that the majority of seminiferous tubules contained a complete complement of germ cell generations, including elongate spermatids. However, the degree of synchrony observed was reduced with respect to previous groups. All animals were synchronized between stages IX-IV of the cycle of the spermatogenic epithelium. However, considerable interindividual variation with respect to the precise location of synchrony was observed, ranging from stages IXXIV to XIV-III. Seventy days after vitamin A replacement, full spermatogenesis was observed in the majority of tubules. However, two animals within this group showed high percentages of tubules with degenerative features. The range of synchronization was much wider than observed in previous groups. However, within individual animals a high degree of synchronization was still observed. The individual range of synchrony varied from stages I—II, XII—II, and VI-XIV in different animals. Overall the mean ratio of synchrony was lower than in previous groups. Eighty-four days post vitamin A injection, many tubules contained large proportions of degenerating round spermatids. However, many such tubules were classified due to the presence of type B spermatogonia as being from stages V-VII. In addition, many tubules containing type B spermatogonia or type B spermatogonia and pachytene spermatocytes were also observed. At this time five out of six animals displayed synchrony between stages V-VII, with a single animal displaying synchrony between stages I-V of the cycle of the seminiferous epithelium. Ninety-eight days after replacement of vitamin A, many tubules showed once again severe regression with a single animal showing 99% Sertoli cell only tubules. In the remaining five animals within this group varying degrees of synchronization were observed. Two animals

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754

GROWTH FACTORS IN SYNCHRONIZED SPERMATOGENESIS

showed synchrony between stages V-VII, two between stages XI-I and a single animal showed a slightly biased distribution of stages with increased numbers of tubules at stage I of the cycle of the seminiferous epithelium. However, this animal also displayed large numbers of damaged tubules. One hundred and fifteen days after restoration of vitamin A, all animals displayed synchrony between stages V-VIII of the cycle of the seminiferous epithelium. Significant proportions of tubules did not contain elongate spermatids. Otherwise, spermatogenic function was normal in these animals. The degree of synchrony observed, however, was further reduced in comparison with previous groups. One hundred and nineteen days post vitamin A injection, spermatogenesis was synchronized to a variable degree in all animals. Four animals showed synchrony between stages VII-XII, one between stages X-III and the final animal between stages I-V of the cycle of the seminiferous epithelium. One hundred and twenty-two days after resupplementation of vitamin A, a very variable pattern of synchrony was observed. Two animals were synchronized between stages V-VIII, two between stages IX-XIV, one between stages IX-II, one between stages XII-IV and one between stages I-VII. In addition, increasing numbers of degenerating tubules were observed within this group. Finally, 126 days post injection of vitamin A, two animals were synchronized between stages V-VIII, two between stages I-VIII, and two animals showed more than 90% of tubules containing spermatogonia and Sertoli cells or Sertoli cells only. Ratio of synchrony

After vitamin A treatment, a ratio of synchrony was estimated for all animals, as described above, and overall this index of synchrony decreased markedly with time (Fig. 3). However, when individual values are plotted, a marked variation between animals in the degree of synchrony observed existed. Calculations based on linear regression of the values for the ratio of synchrony suggest that within 35-40 weeks post vitamin A treatment, a return to a normal distribution of stage frequency within the cycle of the seminiferous epithelium could be expected. Discussion The role of vitamin A in the maintenance of normal testicular function has been widely studied. Vitamin A deficiency in rats results in growth arrest and spermatogenic regression to preleptotene stage, with elevation of serum FSH (29-31). In vitro studies have also implicated vitamin A in the regulation of growth factor pro-

5.01

Endo'1990 Vol 127 «No 2

= -0.108 x + 3.83 r = 0.90

4.03.0

2.0

1.0 0.0

1 5.01 4.0-1

3

5

7

9

11

13

15

17

19

15

17

19

Time (weeks) post treatment y = -0.095 x +3.65 r = 0.66 O

Q

3.0

2.01.0 0.0

5

7

9

11

13

Time (weeks) post treatment FIG. 3. Decrease in the ratio of synchrony in animals displaying synchronized spermatogenesis with time. Regression analysis of the mean ratio of synchrony {upper panel) and of the ratio of synchrony from individual animals (lower panel) against time. A ratio of synchrony was calculated as follows: The stages over which synchrony was observed were classified as those stages showing at least 10% of all classified tubules, or inclusive of stages lying between two such stages. The percentage sum of tubules represented within synchronized stages under this classification was then divided by the percentage sum of tubules present within such stages in control animals to produce a ratio reflecting the degree of synchrony achieved. This value was then analyzed with respect to time.

duction within the testis. Synergistically with FSH, retinol stimulates the production of an EGF-like factor from mouse cryptorchid testes which has been implicated in the regulation of spermatogonial division (2-4). Sertoli cell production of seminiferous growth factor (32) is also regulated by retinol (33). In addition submandibular gland EGF appears to be essential for spermatogenesis in the mouse (34). The data presented here show that at stages IX-XIV of the cycle of the seminiferous epithelium, testicular EGF concentrations are significantly higher than at other stages of the spermatogenic cycle. Stages IX, XII, and XIV of the cycle of the seminiferous epithelium represent the stages where the three mitotic divisions of type A spermatogonia occur in the rat (35, 36). Therefore the data presented here provide the first in vivo evidence of a correlation between EGF or an

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GROWTH FACTORS IN SYNCHRONIZED SPERMATOGENESIS EGF-like factor and the regulation of spermatogonial cell division in the rat. Should this phenomenon be confirmed in further studies, it would represent the first established link between germ cell divisions and growth factor production within the testis. Rat EGF differs from human and mouse EGF in that it contains 4 fewer amino acids [49 us. 53 amino acids (37)]. We have employed a human antiserum to measure rat EGF secretion within the testis and have validated the human EGF assay for measurement of rat EGF using a purified rat EGF standard. Using two separate mouse anti-EGF antisera, no cross-reactivity between mouse and rat EGF could be demonstrated (data not shown). Previous data (37) suggest that murine and rat EGF are biochemically dissimilar and that biochemical studies using murine EGF to compare with rat EGF should be interpreted with caution. Recently data has been presented suggesting that the EGF-like factor produced within the testis is distinct from murine EGF (38). Data presented here would suggest, however, that authentic rat EGF measured with an antiserum which does not cross-react with rat TGF-« (Amersham, Braunschweig, F.R.G.) is present within the testis. Our observations suggest that comparisons between murine and rat EGF can lead to erroneous conclusions. Although previous studies have demonstrated that secretion of a testicular EGF-like material is stimulated by retinol, no conclusive evidence of this phenomenon was observed in the current experiment. However, testicular EGF concentrations are higher in animals immediately after vitamin A treatment than in deficient animals, although this difference is not statistically significant. The possibility exists that retinol stimulates EGF secretion in the present study but that such an effect is masked by other regulators of testicular EGF production. In the current study, two sub-groups have been identified in animals synchronized between stages IX-XIV. In the subgroup where secondary spermatocyte numbers are reduced or absent, significantly higher testicular EGF concentrations are present as compared to the subgroup synchronized around the same stages where secondary spermatocytes are present. While caution is required in interpreting this data, these findings would appear to constitute supportive evidence for a role of EGF in the maintenance of secondary spermatocyte function. However, an alternative explanation may also be that retinol stimulation of animals previously deficient in this substance results in stimulation of testicular EGF and since animals recovering from retinol deficiency lack secondary spermatocytes these phenomena, while not causually linked, occur simultaneously. Further experimentation with regard to testicular EGF and its relationship to the status of secondary spermatocytes within the testis is required. However, the correlation of type A spermato-

755

gonial division with elevated testicular EGF concentrations occurs temporally distant from injection with retinol (8-19 weeks post injection) suggesting that this phenomenon relates to a physiological regulation of testicular EGF. After replenishment of vitamin A in deficient animals, a highly significant rise in testicular IGF-I concentrations was observed. In vitamin A-deficient animals, before vitamin A injection, testicular IGF-I concentrations were approximately 60% higher than in normal control animals. After injection of vitamin A, IGF-I concentrations in both previously untreated controls and vitamin A-deficient animals rose markedly (by 60% and 140%, respectively). Although levels in control animals fell by 28 days post treatment to concentrations comparable to pretreatment levels, throughout the observation period, testicular IGF-I levels remained elevated in control animals by 2080%. In treated animals, testicular IGF-I concentrations remained elevated by more than 140% up to 42 days post vitamin A injection. Levels fell to pretreatment levels by 56 days post vitamin A treatment. However, at all time points throughout the period of observation, testicular IGF-I concentrations remained markedly and significantly elevated above control concentrations. No such changes in serum IGF-I concentrations were observed. IGF-I is produced in many different tissues including liver, lung, kidney, and testes, all of which contain similar concentrations of IGF-I (7). It would appear that the regulation of IGF-I production in different tissues is markedly different. Liver IGF-I production, which accounts for circulating IGF-I, is dependent largely upon GH (11, 39-40) while testicular production of IGF-I or its mRNA is also stimulated by FSH and LH (41). In addition, when animals were stimulated with FSH and LH, no increase in serum IGF-I was observed despite an increase in testicular concentrations of this peptide (41). No changes in serum or pituitary GH concentrations were evident in vitamin A-deficient animals before or after resubstitution with vitamin A. This observation could account for the lack of effect of vitamin A on circulating IGF-I concentrations. Furthermore, the data support the concept that GH probably does not play a major role in the regulation of testicular IGF-I concentrations since marked changes in testicular IGF-I concentrations occurred throughout the study period despite no changes in serum or pituitary GH concentrations. In addition, it is suggested that retinol plays a major role in the regulation of testicular IGF-I production since in both vitamin A-deficient and control animals, administration of retinol produced a marked increase in testicular concentrations of IGF-I. FSH has been shown to stimulate testicular IGF-I mRNA production (41), and retinol stimulation of vitamin A-deficient animals results

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GROWTH FACTORS IN SYNCHRONIZED SPERMATOGENESIS

in a restoration of normal FSH receptor concentrations within the testis (33). However, this phenomenon does not explain the marked stimulation of testicular IGF-I observed in control animals, in which FSH receptors are present in normal concentrations, after retinol injection. Therefore it would appear that the stimulatory effect of retinol on testicular IGF-I is a direct and not an indirect effect mediated, for example, by an effect on FSH receptor concentrations. Retinol binding proteins are localized on both Sertoli cells and Leydig cells within the testis (42, 43). Therefore direct stimulation of IGF-I production or modulation of gonadotropin stimulation of these cells by retinol is feasible. IGF-I receptors are localized on Leydig cells, Sertoli cells, and pachytene spermatocytes (8, 14, 20). Stimulation of immature Sertoli cells with IGF-I in culture increases thymidine incorporation, glucose uptake, and lactate production in these cells (18,19). IGF-I and FSH synergistically increase FSH-stimulated cAMP production and plasminogen activator secretion (17). Glucose uptake, lactate, and plasminogen activator have all been suggested to play a role in maintaining secondary spermatocyte function. Increased testicular IGF-I, which increases production of these paracrine factors in vitro, could therefore reflect increased Sertoli cell activity in situations in which secondary spermatocyte function is compromised or during recovery of spermatogenic function. Increased IGF-I production around stages VII-VIII, where an increase in metabolic activity has been reported (44), is suggested by some data presented here, but the data cannot be regarded as conclusive. However, conclusive evidence relating to retinol stimulation of testicular IGF-I and dissociation of testicular and serum IGF-I concentrations is presented here. This data substantiate previous reports that serum and tissue IGF-I concentrations do not correlate (45). Both EGF and IGF-I have been implicated in the regulation of Leydig cell testosterone production (4650). However, no correlation between testicular testosterone concentrations and growth factor concentrations could be demonstrated in the present study. Although both IGF-I concentrations and testicular testosterone concentrations were markedly stimulated by vitamin A treatment in treated animals, testicular IGF-I concentrations fell before testicular testosterone levels. In addition, although testicular IGF-I concentrations did not normalize during the course of this study, testicular testosterone concentrations returned to levels not significantly different from controls 56 days after vitamin A treatment. Numerous factors have already been implicated in the regulation of Leydig cell steroid production (51, 52), and it is improbable that such effects, even if physiologically relevant, will be demonstrable in in vivo studies. Stage synchronization after withdrawal of vitamin A

Endo • 1990 Voll27«No2

and subsequent replenishment was first reported in 1987 (22). After this first communication, several studies have been published relating to the usefulness of this model for the analysis of testicular function (23, 24). The data on stage synchronization with respect to time presented in this study suggest that the degree of synchrony decreases with time. However, due to large interindividual variation observed, no definite conclusion can be drawn as to the stability of synchrony induced by vitamin A withdrawal. From data included here, it would appear that one problem is the relatively short duration of studies published to date. Our data suggest that for full desynchronization of spermatogenesis to be achieved, a period of between 35-40 weeks after initial replacement of vitamin A would be required (calculated from regression curves). Should this indeed be the case, further studies of longer duration should enable this point to be finally addressed. However, although the evidence that such desynchronization occurs is growing, it is not possible to definitely conclude that variation in the temporal duration of individual stages of the cycle of the seminiferous epithelium as originally observed by Leblond and Clermont (28) contributes to a progressive desynchronization of spermatogenesis in this model. To date studies of stage synchronization in which testicular wts have been reported have established that after vitamin A deficiency and subsequent synchronous development of spermatogenesis, only partial recovery in testicular function, as determined by testicular wt and quantitative histological examination (24), can be achieved. Similar findings are presented here. In vitamin A-deficient rats, a marked reduction in testicular size is observed (45% of control values). Immediately after vitamin A injection, testicular wts decline further before recovering to reach a maximum of 65% of control testicular wts. It would appear that induction of retinol deficiency has marked and permanent deleterious effects on testicular function and perhaps the organism as a whole. Epididymal wts have not yet been shown to recover after vitamin A deficiency (Ref. 24 and above). However pituitary wts do reach normal levels after replenishment of vitamin A. Both serum and pituitary gonadotropin concentrations showed changes similar to those reported previously (24). It is of interest that although testicular wts never normalize, serum FSH concentrations remain normal between 42 and 119 days post vitamin A treatment, the period at which testicular wts recover to their maximal values in treated animals. It has been observed that marginal testicular damage, involving less than 30% of testicular mass, does not result in elevation of serum FSH (53, 54), and it is possible that the feedback control of FSH is sufficient in these animals to normalize serum levels of this hormone. This hypothesis is supported by

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GROWTH FACTORS IN SYNCHRONIZED SPERMATOGENESIS

the subsequent rise in FSH values observed between 119-126 days post treatment when testicular wts and testicular morphology decline. The data presented here show that after vitamin A withdrawal and replenishment major changes in the regulation of testicular growth factor secretion can be demonstrated. After synchronization of the cycle of the seminiferous epithelium, significantly higher concentrations of EGF were observed at stages IX-XIV of the cycle of the spermatogenic epithelium than at stages V-VIII. Few animals synchronized around stages I-IV could be observed, but in five animals synchronized between stages I—VIII, testicular EGF concentrations were also lower than at stages IX-XIV. This data support in vitro evidence (2-4) that EGF may be involved in the regulation of type A spermatogonial divisions occurring at stages IX, XII, and XIV. Data relating to testicular concentrations of IGF-I suggest that this factor may be regulated by retinol. No stage-dependent changes in IGF-I concentrations could be demonstrated.

Acknowledgments

13.

14. 15. 16. 17.

18. 19. 20. 21.

The authors would like to thank J. Esselmann, M. Heuermann, R. Sandhowe, and G. Stelke for technical assistance. 22.

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EC, Lund PK 1987 Isolation of rat testis cDNAs encoding an insulin-like growth factor I precursor. DNA 6:325 Hernandez ER, Roberts Jr CT, LeRoith D, Adashi EY 1989 Rat ovarian insulin-like growth factor I (IGF-I) gene expression is granulosa cell-selective: 5'-untranslated mRNA variant representation and hormonal regulation. Endocrinology 125:572 Tres LL, Smith EP, Van Wyck JJ, Kierszenbaum AL 1986 Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures. Exp Cell Res 162:33 Vanelli BG, Barni T, Orlando C, Natali A, Serio M, Balboni GC 1986 Insulin-like growth factor-I (IGF-I) and IGF-I receptor in human testis: an immunohistochemical study. Fertil Steril 49:666 Hansson HA, Billig H, Isgaard J 1989 Insulin-like growth factor I in the developing and mature rat testis: immunohistochemical aspects. Biol Reprod 40:1321 Saez JM, Chatelain PG, Perrard-Sappori M-H, Jaillard C, Naville D 1988 Differentiating effects of somatomedin-C/insulin-like growth factor I and insulin on Leydig and Sertoli cell functions. Reprod Nutr Dev 28:989 Borland K, Mita M, Oppenheimer CL, Blinderman LA, Massague J, Hall PF, Czech MP 1984 The actions of insulin-like growth factors I & II on cultured Sertoli cells. Endocrinology 114:240 Mita M, Borland K, Price JM, Hall PF 1985 The influence of insulin and insulin-like growth factor-I on hexose transport by Sertoli cells. Endocrinology 116:987 Oonk RB, Grootegoed JA 1988 Insulin-like growth factor-I (IGFI) receptors on Sertoli cells from immature rats and age dependent testicular binding of IGF-I and insulin. Mol Cell Endocrinol 55:33 Adashi EY, Resnick CE, Hernandez ER, May JV, Knecht M, Svoboda ME, van Wyk JJ 1988 Insulin-like growth factor I as an amplifier of follicle stimulating hormone action: studies on mechanism^) and site(s) of action in cultured rat granulosa cells. Endocrinology 122:1583 Morales C, Griswold D 1987 Retinol-induced stage synchronization in seminiferous tubules of the rat. Endocrinology 121:432 Morales CR, Acacia AA, Hecht NB, Griswold MD 1989 Specific mRNAs in Sertoli and germinal cells of testes from stage synchronized rats. Mol Endocrinol 3:725 Bartlett JMS, Weinbauer GF, Nieschlag E 1989 Quantitative analysis of germ cell numbers and relation to intratesticular testosterone following vitamin A induced sychronization of spermatogenesis in the rat. J Endocrinol 123:403 Kreysing P, Nieschlag E 1986 Direct chemiluminescence assay for testosterone. J Biolum Chem 1:109 Bartlett JMS, Weinbauer GF, Nieschlag E 1989 Differential effects of FSH and testosterone on the maintenance of spermatogenesis in the adult hypophysectomized rat. J Endocrinol 121:49 Daughaday WH, Mariz IK, Blethen SL 1980 Inhibition of access of bound somatomedin to membrane receptor and immunobinding sites: a comparison of radioreceptor and radioimmunoassay of somatomedin in native and acid-ethanol extracted serum. J Clin Endocrinol Metab 51:781 Leblond CP, Clermont Y 1952 Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci 55:548 Mason KE 1933 Differences in testes injury and repair after vitamin A deficiency, vitamin E deficiency and inanition. Am J Anat 52:153 Huang HFS, Hembree WC 1979 Spermatogenic response to vitamin A in vitamin A deficient rats. Biol Reprod 21:891 Huang HFS, Dyrenfurth I, Hembree WC 1983 Endocrine changes associated with germ cell loss during vitamin A-induced recovery of spermatogenesis. Endocrinology 112:1163 Bellve AR, Feig LA 1984 Cell proliferation in the mammalian testis: biology of the seminiferous growth factor. Recent Prog Horm Res 40:531 Unni E, Rao MRS 1986 Androgen binding protein levels and FSH binding to testicular membranes in vitamin A deficient rats and during subsequent replenishment with vitamin A. J Steroid Biochem 25:579 Tsutsumi O, Kurachi H, Oka T 1986 A physiological role for epidermal growth factor in male reproductive function. Science 233:975

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35. Clermont Y 1972 Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 52:196 36. Clermont Y, Harvey SC 1965 Duration of the cycle of the seminiferous epithelium of normal, hypophysectomized and hypophysectomized-hormone treated albino rats. Endocrinology 76:80 37. Schaudies RP, Savage CR Jr 1986 Isolation of rat epidermal growth factor (r-EGF): chemical, biological and immunological comparisons with mouse and human EGF. Comp Biochem Physiol 84:497 38. Skinner MK, Takacs K, Coffey RJ 1989 Transforming growth factor-a gene expression and action in the seminiferous tubule: peritubular cell-Sertoli cell interactions. Endocrinology 124:845 39. Hynes MA, Van Wyk JJ, Brooks PJ, D'Ercole AJ, Jansen M, Lund PK 1987 Growth hormone dependence of somatomedin-C/insulinlike growth factor I and insulin-like growth factor II messenger ribonucleic acids. Mol Endocrinol 1:233 40. Mathew LS, Norstedt G, Palmiter RD 1986 Regulation of insulinlike growth factor I gene expression by growth hormone. Proc Natl Acad Sci USA 83:9343 41. Closset J, Gothot A, Sente B, Scippo ML, Igout A, Vandenbroeck M, Dombrowicz D, Hennen G 1989 Pituitary hormones dependent expression of insulin-like growth factors I and II in the immature hypophysectomized rat testis. Mol Endocrinol 3:1125 42. Cope FO, Knox KL, Hall RC 1984 Retinoid binding to nuclei and microsomes of rat testes interstitial cells 1. Mediation of retinoid binding by cellular retinoid-binding proteins. Nutr Res 4:289 43. Kato M, Kong Sung W, Kato K, Goodman DS 1985 Immunohistochemical studies on the localization of cellular retinol-binding protein in rat testis and epididymis. Biol Reprod 32:173 44. Parvinen M 1982 Regulation of the seminiferous epithelium. En-

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