Exp. Clin. Endocrinol. 99 (1992) 49-53

Experimental and Clinical Endocrinology

A Delayed Response to Acute Stimulation with Human Chorionic Gonadotropin in Scorbutic Rats Y. Ito'), M. Tsuji'), M. Miyano'), K. Wakabayashi2) and H. Mon') Department of Pathology') (Director: Prof. Dr. H. Mon), Osaka Medical College, Takatsuki/Japan and Institute of Endocrinology2) (Director: Prof. Dr. K. Wakabayashi), Ounma University, Maebashi/Japan

Key words: Testosterone - LH/hCG - Scurvy - Ascorbic acid - ODS rats Summary: Since our previous study revealed low basal testosterone (T) levels and a failure in response to an acute stimulation with human chorionic gonadotropin (HCG) despite of a good response to chronic stimulation in scorbutic mutant rats, time course of the response to HCG was studied and plasma LH level was measured in young adult rats deficient in ascorbic acid for 3

weeks. A single subcutaneous injection of HCG (200 lU)

elevated T levels only slightly in plasma and not in testicular tissues of scorbutic rats 1 h after the injection when the levels in ascorbutic rats reached a maximum, while it yielded the same response pattern as in ascorbutic rats after 3 h. A pretreatment with HCG to scorbutic rats for 1 or 4 days resulted in the same response of plasma T as in ascorbutic rats. Plasma LH levels in unstimulated scorbutic rats were about 400/ of those in ascorbutic rats. These findings indicate that a prolonged deficiency of ascorbic acid decreases plasma LH, and may reduce the sensitivity of testes to LH.

Introduction

have been reported so far (Björkhem et al., 1978; Hodges and Hotston, 1970; Gombe et al., 1977). The reason may

Ascorbic acid (AsA) is contained in steroid hormonesecreting tissues at high concentrations (Hornig, 1975).

be ascribed partly to the difficulty in producing the

Findings that an injection of ACTH or LH causes a depletion of AsA in adrenal glands or corpora lutea with a

concomitant increase in plasma levels of glucocorticoid (Lipscomb and Nelson, 1960) or progesterone (Hokfelt,

1949) have established bioassay methods for ACTH (Sayers et al., 1948) and LH (Parlow, 1958). AsA has been considered to play an important role in the produc-

tion and secretion of steroid hormones (Levine and Monta, 1958). However, numerous studies, mostly in vitro using the adrenal tissues, have shown complicated results concerning the role of AsA in the steroidogenesis

(Tsuji et aI,, 1989; Tsuji et al., 1990; Ito et al., 1990; Fukuda et al., 1990; Fukuda et al., 1991): AsA either stimulates some steroidogenic enzymes, inhibits other enzymes, or has no effect on others according to enzymes

scorbutic state in laboratory animals except for guinea pigs and primates because of the ability of endogenous AsA production. ODS (Osteogenic disorder Shionogi) rats, recently established mutant rats, can not synthesize AsA because of the lack of l-gulonolactone oxidase that catalyzes the final step of AsA biosynthesis, and become scorbutic when not supplied with dietary AsA (Konishi et al., 1990).

Previously we reported using the ODS rats that a deficiency of AsA resulted in a difference in vivo production of steroids between the adrenal cortex and the testis (Fukuda et al., 1991). Corticosterone (C) levels in plasma

and adrenal tissues of scorbutic rats were higher than those in ascorbutic rats. A circadian change of plasma C

was maintained in both ascorbutic and scorbutic rats. Acute and chronic stimulations with ACTH elevated C

examined and conditions employed. The in vivo study

levels in both ascorbutic and scorbutic rats. It seemed that

seems more informative on the physiological role of AsA than the in vitro study. However, only few in vivo studies

the scurvy elevated plasma ACTH levels secondary to the stress, resulting in a stimulation of the adrenals. On

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© 1992 Johann Ambrosius Barth

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A chronic stimulation with HCG elevated T levels remarkably in both ascorbutic and scorbutic rats. However, an acute stimulation elevated T levels only slightly in plasma and not in testicular tissues of scorbutic rats when T levels in ascorbutic rats were elevated remarkably. We wondered if a prolonged deficiency of AsA might decrease plasma gonadotropin levels, and reduced the sensitivity of testes to gonadotropins. To clarify this assumption, the present study measured plasma LH levels and examined a time course of the response to HCG in scorbutic and ascorbutic ODS rats.

Measurement of LH

Rat LH in plasma was measured by double antibody radioimmunoassay using NIDDK RIA kit (NIDDK rLH-I-7, anti-rLHS-11 and rLH-RP-3) supplied by National Hormone and Pituitary Program (Baltimore, MD, USA), and a goat anti-rabbit IgG serum (HACRBA2-03GTP86) supplied by Hormone Assay Center, Institute of Endocrinology, Gunma University (Maebashi, Gunma, Japan), according to the instruction paper issued by NIDDK with a minor modification (Wakabayashi, 1977). Intra- and inter-assay coefficients of variation were less than 10%. Statistical analysis

The data were subjected to statistical analysis using Student's t test.

Results

Materials and Methods Animals and diet

Male ODS rats (ODS/Shi-od/od) were provided from Aburahi Laboratories, Shionogi and Co., Ltd. (Shiga, Japan). Conditions of breeding were almost the same as in previous study

(Fukuda et al., 1991). Briefly, all rats were fed on an AsAdeficient lab chow of CL-2 (Clea Japan Inc., Tokyo) after the experiments started at the age of 28 days. The lab chow was superheated at 121 °C for 7 min just before use to ensure the destruction of AsA. The AsA content in the lab chow thus treated was below 1 mg/lOO g (5.7 smo1/l00 g; minimum

Basal T levels in plasma and testicular tissue of unstimu-

lated scorbutic rats [ODS(-) rats] in the morning were about half and one-third the values in AsA-supplied rats [ODS(+) rats], respectively (Fig. 1), Plasma T levels in

the night elevated in ODS(+) rats but not in ODS() rats. In testicular tissues T concentrations in the night

detectable concentration). One group of rats was supplied with

AsA, by giving drinking water containing 0.1% AsA, and served as a control. Rats were maintained under controlled temperature (23 ± 2°C) and lighting (light on 06:00-18:00), and supplied with food and water ad libitum. At the end of experiments, rats were sacrificed by decapitation without giving any stress or stimulation as carefully as possible. The plasma

10

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E (I)

(0

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and testes were stored at 20 or 80 °C for the measurement of T and/or LH. For the time course study of an acute stimulation with HCG, rats kept unstimulated for 3 weeks were injected sc with 200 lU of HCG (Mochida Pharmaceut. Co., Tokyo) on the 22nd day,

and decapitated 1, 3, 6, 9, 12 and 24h later. To examine the effect of HCG pretreatment, rats kept unstimulated for 20 and 17 days were injected sc with 200 lU of HCG once a day for 1 and 4 days, respectively, and sacrificed 1 and 3 h after the final, additional injection on 22nd day.

Measurement of T

T levels in plasma and testicular tissues were determined by radioimmunoassay as described previously (Fukuda et al., 1991). Steroids in 0.05-0.1 ml of the plasma or the testicular homogenates were extracted by 4m! of distilled methylene chloride. The extracted steroids were incubated at 37 °C for 15 min ïn 0.3 ml of the solution containing anti-testosterone antiserum and 3H-testosterone (106 dpm). The antibody raised against testosterone-3-carboxymethyloxime-bovine serum albumin was generously supplied by Dr. H. Imura, Kyoto Univer-

I

(0

e o, 0.1 E C,)

*

0.05

ODS()

ODS(-)

Fig. 1 Testosterone levels in unstimulated rats. Male ODS rats were kept unstimulated on an AsA-deficient lab chow and given drinking water containing [ODS(+)] and not containing [ODS()I AsA for 3 weeks. They were decapitated at 08.30 h

and 20.30h on 50 days of age. The levels in plasma and testicular tissue of ODS () rats were less than half the values of

ODS(+) rats both in the morning (1

1) and in the night

sity School of Medicine. [1, 2, 6, 7-3H] testosterone (88 Cil

). The plasma levels in the night were elevated in ODS (+) rats but not in ODS () rats. Values are mean ± SEM (N = 5).

mmol) was obtained from the New England Nuclear Co. (Bos-

Significant difference (P < 0.05); * from the value in the

ton, MA, USA). The intra- and inter-assay coefficients of

morning in the same rat group, and * from ODS (+) rats at the same hour, respectively

variation were 8.5 and 10.9%, respectively.

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the contrary, testosterone (T) levels in plasma and testicular tissues of unstimulated scorbutic rats were lower than those in ascorbutic rats. A circadian change of plasma T was observed in ascorbutic rats but not in scorbutic rats.

Exp. Clin. Endocrinol. 99 (1992) 1

Y. Ito et al., Response to HCG in Scorbutic Rats

decreased in ODS(+) rats but were still significantly higher than those in ODS( -). When stimulated with a single sc injection of HCG, T levels in ODS(+) rats increased remarkably 1 h after the injection (Fig. 2);

51

assay made 1 h after the injection. To examine whether

the failure in elevation of T levels in unstimulated ODS() rats after a single dose of HCG was indeed due to the weak response, the time course was studied. In ODS(+) rats, plasma T levels were elevated sharply 1 h after an injection of HCG, and decreased at 3 h after, but were still maintained to be high by 12 h after the injection

(Fig. 3). In ODS(

rats, however, T levels were ele-

o E

*

E q,

CO

Q-

0.6

o E

C

(li Q)

0.2

0.5

o

ODS(+)

ODS()

Fig. 2 Testosterone levels responding to acute and chronic stimulation with HCG. Animals were the same as in Fig. 1. They were either unstimulated (Ti), injected with a single dose of HCG (200 lU) on the 22nd day or injected for 22

days (), and sacrificed 1h after the final injection. An acute stimulation elevated testosterone levels remarkably in plasma and testicular tissue in ODS(+) rats, while only slightly in

0.4

o E

C

0.3

Ci)

u)

0.2

0.1

plasma and not at all in the testicular tissue in ODS () rats. The chronic stimulation succeeded in elevating plasma and testicular levels in both rats. Values are mean ± SEM (N = 5). Significant difference (P < 0.05); * from other experimental condition in the same rat group, and * from ODS (+) rats in the same experimental condition, respectively

Fig. 3 Time course of response of testosterone levels to HCG

about 15 times higher in plasma and 4 times higher in

testosterone levels in ODS(+) rats (O-----O) reached a max-

testicular tissues than those of unstimulated ODS(+) rats.

However, T levels in ODS() rats were elevated in plasma only 2 times to those in the unstimulated ODS()

rats, and remained unchanged in testicular tissues. A chronic stimulation elevated T levels in both groups of rats. In ODS(+) rats, although the extent of elevation in plasma T levels was smaller than that in acute stimulation, testicular T levels increased noticeably, similarly to the acute stimulation. In ODS() rats, T in both plasma and the testicular tissue elevated to the levels similar to those in acutely stimulated ODS(+) rats. Results obtained above appeared to indicate that the

o

Time after the hCG injection (h) in plasma (top) and testicular tissue (bottom). Animals were the same as in Fig. 1. They were kept unstimulated for 3 weeks and injected with a single dose of 200 lU HCG on the 22nd day. The

imum 1h after the injection, while those in ODS() rats .) reached the maximum 3h after in plasma and 6h after in testicular tissue. The levels thereafter were the same in both

rats. Each point represents mean ± SEM (N = 5). * Signifi-

cantly different (P < 0.05) from ODS(+) rats. (Figure for plasma level was cited from Reference 11.)

vated only slightly 1 h after the injection. The levels increased thereafter and showed a similar pattern as in ODS(+) rats since 3 h after the injection. Changes in testicular tissue showed a similar trend to those in the plasma, though a little different. T levels in ODS(+) rats

deficiency in AsA for 3 weeks caused a very weak

reached a maximum 1 h after the injection, whereas those

response of rat testis to the acute stimulation with HCG, and that a chronic pretreatment with HCG restored a good response. However, measurement of T was a one-point

in ODS() rats reached the maximum at last 6h after, to the same level as in ODS(+) rats. Changes thereafter were the same in both groups of rats.

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C CO

52

Exp. Clin. Endocrinol. 99 (1992) 1

r*

ODS(+) rats after the pretreatment for 1 and 4 days. It seems to represent HCG-induced desensitization that the

o

levels in ODS(+) rats after 1-day pretreatment were

E

lower than those in ODS(+) rats receiving no pretreatment at 1 and 3 h after the injection. Concentrations of plasma LH in unstimulated ODS()

a) C

o

a) U)

o

rats were lower, about 40% of those in ODS(+) rats

U)

(Fig. 5). Discussion

b

T E

Our previous report (Fukuda et al., 1991) gave rise to questions why T levels in ODS() were lower than in ODS(+) rats, and why the testes of ODS() rats failed in

30

a

0)

20

responding to an acute stimulation with HCG in contrast to ODS(+) rats. The present study revealed that a failure of response to an acute stimulation with HCG in ODS() rats 1 h after the injection of HCG was owing to a delay

a) 10 U)

iday

Oday

4 days

Fig. 4 Effect of HCG pretreatment on the response of plasma testosterone to HCG stimulation. Animals were the same as in Fig. 1. The rats kept unstimulated for 21, 20 and 17 days were injected Sc with 200 lU HCG once a day for 0, and 4 days, respectively, and sacrificed I h (top) and 3 h (bottom) after the 1

final, additional injection on 22nd day. When pretreated for

more than 1 day, ODS() rats () gained a prompt response at 1 h after the stimulations, as in ODS(+) rats (EJ). There was no difference in plasma testosterone levels between

but not a lack of the response. As shown in Fig. 3, T levels in ODS() rats remained almost unchanged at 1 h after the HCG injection, when the levels in ODS(+) rats reached a maximum. However, they elevated remarkably to the same levels as in ODS(+) rats 3 h after in plasma and 6 h after in testicular tissue. The pretreatment with HCG for more than 1 day recovered the same response

pattern to HCG in ODS() rats as in ODS(+) rats. The cause for low T levels in unstimulated ODS() rats and

ODS() and ODS(+) rats after pretreatment for I and 4 days.

the delay of response to HCG appears to be attributed to

A smaller extent of response in ODS (+) rats receiving pretreatment appears owing to desensitization by pretreatment. Values are mean ± SEM (N = 5). Significantly different (P

A delayed response to acute stimulation with human chorionic gonadotropin in scorbutic rats.

Since our previous study revealed low basal testosterone (T) levels and a failure in response to an acute stimulation with human chorionic gonadotropi...
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