Photoperiodically induced variation in testicular RNA, DNA and protein content in hamsters and ground squirrels

Comp. Biochera. Physiol.. Vol. 631[ pp, 363 to 367

0305-0491/79/0107-0363502.00/0

(~ Pergamon Press Ltd 1979. Printed in Great Britain

P H O T O P E R I O D I C A LLY I N D U C E D VARIATION IN TESTICULAR RNA, DNA A N D PROTEIN CONTENT IN HAMSTERS A N D G R O U N D SQUIRRELS SPENCER D. GREENDYKE, JOHN L FREHN, DANIEL S. H. L1u and ARLAN G. RICHARDSON Departments of Biology and Chemistry, Illinois State University, Normal, IL 61761, U.S.A. (Received 4 December 1978)

Abstraet--l. The total testicular content of RNA, DNA and protein was found to decrease sharply in hamsters with shortened photoperiod and in ground squirrels during the spring breeding season. 2. RNA and DNA per g testes were found to increase in both animals, while protein per g testes remained fairly stable. 3. Cell-free protein synthesis by testicular PMS during testicular regression remained constant when expressed per mg of testicular RNA, but decreased 75% when expressed per testes. 4. These findings suggest that decreases in testicular protein synthesis are due to a decrease in RNA content and not to alteration of the translational activity of the RNA.

INTRODUCTION

Seasonal reproductive cycles have been observed for many different species of mammals. For example, reproductive periodicity among certain hibernating male animals is generally characterized by a peak in sexual maturity during the spring, followed by rapid testicular regression (McKeever, 1963). These observations of seasonal reproductive activity of rodents were reported initially for the ground squirrels Citellus trideceralineatus (Wells, 1935) and, more recently, for Citellus beidingi (McKeever, 1963). Similar investigations with male chipmunks, Tamias striatus (Neff & Anthony, 1967), have demonstrated that the initiation of testicular regression occurs between April and May, and testicular size reaches a minimum in the month of June. Numerous studies have been undertaken to elucidate the role of seasonal and environmental factors in triggering reproductive cycles Many of these studies have used golden hamsters. These animals are normally continuous breeders under laboratory conditions (Hoffman et al., 1965); although, some decline in fertility during the winter months has been observed (Deane & Lyman, 1954; Deansley, 1938). The factors which have been found to noticeably influence gonadal activity in golden hamsters are temperature, photoperiod and hibernation (Frehn & Liu, 1970). However, the actual periodicity in hamsters appears to be independent of the occurrence of hibernation (Smits-Vis & Akkerman-Bellhart, 1967). Hoffman & Reiter (1965) have shown that rapid changes in reproductive function, similar to that observed in seasonal breeders` can be induced in golden hamsters by exposure to a short photoperiod. Gaston & Menaker (1967) demonstrated that adult hamsters required at least 12.5 hr of light per day to maintain spermatogenesis and prevent testicular degeneration. Thus, changing photoperiod is considered to be the most important parameter in sychronizing the breed363

ing period with the appropriate season of the year (Elliot, 1976). Frehn & Liu (1970) confirmed the effect of photoperiod on testicular regression~ however, they found that exposure to cold temperatures also would bring about testicular regression. The decline in testicular function with cold exposure or short photoperiod in hamsters is accompanied by decreases in: wet weight, spermatogenic activity, seminiferous tubule diameter (Frehn & Liu, 1970) and glycogen content (Liu, 1972). Although such gross anatomical and histological changes in testes resulting from seasonal and photoperiodic stimuli have been demonstrated in hamsters and seasonal breeders, little is known about the biochemical changes which must accompany them. The purpose of this investigation was to examine the seasonal variation in testicular ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein content in 13-lined ground squirrels captured both during the breeding period following emergence from hibernation and throughout the period of testicular regression. In addition, identical parameters were examined in golden hamsters placed on two photoperiods, one sufficiently long to maintain the testes in an active state, and one which would induce testicular regression. MATERIALS AND METHODS

Animals

Adult male golden hamsters (Mesocricetus auratus) weighing 70-80 g were purchased from Engle Laboratory Animals, Inc., Farmersburg, Indiana. The animals were caged individually with a small amount of shredded paper for nest building, and were given Purina Laboratory Chow and water ad libitum. After being maintained for 2 weeks on 14 hr of artificial light per day at room temperature (24°C :t: l°C), the hamsters were divided into two groups. The control group was maintained under the initial conditions. The experimental animals were moved to a room

364

SPENCER D. GREENDYKE et al.

with identical conditions except that a photoperiod of 1 hr of light and 23hr of darkness (IL:23D) was provided. Control and experimental animals were sampled after 4, 8 and 12 weeks of exposure (mid-April to mid-July). Adult male 13-lined ground squirrels (Spermophilus trideceralineatus) were collected with Havahart wire traps near Illinois State University during and following the mating season (April-June). Determination of RNA, DNA and protein content of testes Animals were decapitated (ground squirrels within 24 hr after capture), and the testes were immediately removed, trimmed of excess connective tissue and weighed. The testes were then decapsulated, and all remaining tissue minus the capsule was weighed. This tissue was then homogenized in 6 vol (6 ml/g tissue) of buffer with 30 passes of a Potter-Elvehjem homogenizer. Aliquots (I ml) of the homogenate were then precipitated with 3 vol of 1 N HCIO,, and the precipitate was washed sequentially with 3 ml of the following: 2:1 chloroform: methanol, 95~0 ethanol, 0.5 N HCIO,, (two washes) and distilled water. The precipitate then was dissolved in 2 ml of 0.3 N NaOH. Samples were removed for the determination of RNA. DNA and protein 10.3 ml, 0.5 ml and 0.05 ml, respectively). The RNA content was determined by a modification of the Schmidt-Trannhauser procedure, using E~,~o+ 312 (32 #g RNA/ml = 1.00 A2~0nm) as described by Munro & Fleck (1966}. The DNA content was determined by the method of Cerotti (1952), using salmon sperm DNA as a standard. The protein content was determined by the method of Lowery et al. (1951), using bovine serum albumin as a standard. Cell-free protein synthesis Because Richardson et al. (1971) have reported that cellfree protein synthesis by post-mitochondrial supernatant IPMS) of tissue homogenate more closely represents the in t,ivo situation than more purified cell-free protein synthesis systems, testicular protein synthesis was measured by the PMS obtained from the homogenate of the testes. The conditions used for cell-free protein synthesis by the testicular PMS were optimum and similar to those reported by Means et al. (1969) for testicular polyribosomes. Immediately after decapitation of the hamsters testes were weighed, decapsulated, reweighed and homogenized at 4°C with a Potter-Elvehjem tissue homogenizer in a buffer consisting of 75mM KCI, 15mM MgCI 2. 50mM HEPES (N-2-hydroxyethylpiperazine-N'-2'-ethane-sulfonic acid), 3 mM glutathione. 0.25 M sucrose and 20-25 mM KOH to obtain a final pH of 7.3. All subsequent procedures were performed at 4~C. The homogenate was then centrifuged at 20,000 ,q for 10 min. The resulting supernatant, PMS. was then chromatographed on a 1.5 x 13 cm Sephadex G-25 column. Homogenization buffer was used to elute the PMS from the column, and the first 3 ml eluted was used to determine cell-free protein synthesis. Measurement of protein synthetic activity was performed at 37°C

by adding Sephadex chromatographed PMS to an equal volume of the homogenization buffer supplemented with 2 mM ATP, 0.5 mM GTP, 14.8 mM creatine phosphate. 0.2 mg/ml creatine phosphatase, a mixture of L-amino acids minus L-valine (final concentration of each amino acid was 50mM) and l gCi/ml of [aH]L-valine (specific activity 2 Ci/mmol). Under these conditions, the amount of [3H]Lvaline incorporated into acid-precipitable material was directly proportional to the RNA content of the PMS (Liu et al., 1978). Protein synthesis was terminated after 60 rain of incubation by the addition of 5~/~ trichloroacetic acid supplemented with 0.2~o OL-valine. 0.5 mg of bovine serum albumin was added to each sample as a carrier protein. The samples were then heated for 15 min at 90'C to hydrolyze the [3H]L-valine covalently bound to tRNA. The resulting mixtures were then washed twice with 5Y,. trichloroacetic acid supplemented with 6 mM OL-valine. and once with the following solvents: 5~o trichloroacetic acid, 2:1 ethanol:ether (pH 7.0), acetone and ether. The remaining material was dried and suspended in 0.3 N N a O H The radioactivity and RNA content of the NaOH suspension were determined using a liquid scintillation counter and by the method of Cerotti et al. (1952), respectively. The protein synthesis was expressed as the dpm of [3H]L-valine incorporated into acid-precipitable material per mg of PMS RNA, and total dpm of [aH]L-valine incorporated into acid-precipitable material per testes. Statistical analyses Ground squirrel data were analyzed using a one-way analysis of variance; and data for the hamster groups were analyzed by a two-way analysis of variance on unweighted means (Snedecor, 1969).

RESULTS G r o u n d squirrels for this study were collected in April,.May and June. Table I shows the body weights and testicular wet weights observed in these ground squirrels. Although there was no significant change in body weight during this time, there was an 86~,, reduction in total testicular weights, and a 92~,,, decrease in decapsulated weights from April to June. Table 2 shows that similar results were obtained for golden hamsters in which testicular regression was induced by a shortened photoperiod. After 12 weeks of treatment, experimental animals demonstrated a 75% reduction in total testes weight, and a 76°/,i decrease in decapsulated weights. Table 3 shows the effect of season on macromolecular parameters in ground squirrel testes. Significant reductions (P < 0.001) in total RNA, D N A and protein levels were found between the m o n t h s of May and June. In addition, a 36% decrease in the protein;

Table I. The effect of season on testicular wet weights in 13-lined ground squirrels

Month Captured

N

Body w e i g h t (BW)

Testes w e i g h t

Testes w e i g h t minus capsule weight (-C)

-C 8W

April

3

178 +- 8.0

1,57

± 0.18

1.30 _+ 0.13

72.8

May

4

175 _* 6.0

1.40 _+ 0.15

1.23 _+ 0.14

69.7

June

3

166 ± 4.0

0.17 ± 0.01

0.I0 ± 0.01

6.0

All weights are expressed in g _+ S.E.M.

365

Testicular RNA. DNA and protein content in hamsters Table 2. Weight changes in the testes of golden hamsters exposed to control (14 light, 10 dark) and experimental (I light, 23 dark) photoperiods

Week

Body weight (BW)

Testes weight minus capsuls weight (-C)

Testes weight

-C BW

Treatment

N

Control

6

126 ± 2.0

3.86 ± 0.22

3.10 ± 0.07

245

Exper.

4

125 ± 3.0

3.29 ± 0.49

2.95 ± 0.44

236

8

Control

6

133 ± 5.0

3.81 ± 0.12

3.51 ± 0.12

265

Exper.

9

126 ± 6.0

2.16 ± 0.36

1.94 ± 0.33

138

12

Control

3

147 ± 7.0

4.43 ± 0.32

4.03 ± 0.40

278

Exper.

6

126±6.0

1.10±0.46

0.99±0.40

.79

4

All weights are expressed in g + S.E.M. Table 3. The effect of season on testicular RNA, DNA and protein content in 13-linedground squirrels RNA Month Captured

N

Total

DNA Per g

Total

Protein Per g

Protein

Total

Per g

DNA

April

3

0.58±0.05

0.44±0.02

0.84±0.07

0.65±0.08

11.8±1.5

8.9±

0.4

14±2

May

4

0.69±0.04

0.58±0.05

0.87±0.05

0.73±0.08

12.1±0.8

i0.0±

0.6

14±1

June

3

0.07±0.01 a

0.71±0.09

0.13±0.I a

1.32±0.14 a

12.1±

4.2

9±3

1.2±0.4 a

Values are expressed in mg + S.E.M. ~ ( P < 0.001).

DNA ratio was found; however, this difference was not statistically significant. The smallest macromolecular reduction occurred in the total DNA levels, which exhibited a decrease of 85%. The 90% reduction in total testicular RNA and protein content was consistent with the reduction of decapsulated testicular weights observed from April to June. The concentrations of all macromolecules appeared to increase slightly with time when expressed per g testes Moreover, the DNA per g testes actually increased 79~ (P < 0.001) during the month of June. Table 4 shows the effect of short photoperiod upon testicular RNA, DNA and protein content in ham-

sters. After 12 weeks of treatment, total RNA and protein levels decreased 69% and 74%, respectively. However, total DNA levels decreased only 34%. Significant differences were observed between the control and experimental hamsters beginning at 8 weeks, for both total RNA ( P < 0 . 0 5 ) and total protein (P < 0.001). The apparent decrease in total DNA did not prove to be significant, possibly due to a rather small sample number within the 12-week group. No differences were observed in either RNA per g testes or DNA per g testes within any group over time, or between control and experimental hamsters; while the protein per g testes values decreased within the

Table 4. Protein, RNA and DNA content in the testes of golden hamsters exposed to control (14 light, 10 dark) and experimental (1 light, 23 dark) photoperiods DNA Week

4

8

12

Total

Per g

Protein

Treatment

N

Control

6

5.63 ± 0.56

1.83 _+ 0.19

3.09±0.60

1.00±0.20

29.8±2.6

9.7±0.9

Exper.

4

5.12 ± 0.98

1.90 ± 0.51

2.97±0.22

1.12±0.26

27.7±4.4

9.6±1.1

Control

6

6.28 + 0.i0

1.81 ± 0.09

3.23±0,23

0.92±0.06

43.2±2.7

12.3±0.7

Exper.

9

3.57 ± i.I0 c

1.67 ± 0.34

2.44±0.21

1.15±0.17

18.5±3.4 a

Control

3

4.50 ± 1.89

,1.08 ±'0.28

2.78±0.31

0.71±0.I0

46.5±8.5

Exper.

6

1.38 ± 0.47

1.66 _+ 0.64

1.79±0.39

3.11±0.76

ii.9±3.11 a

Values are expressed in mg + S.E.M. a(p < 0.001); b(p < 0.01); ¢(P < 0.05).

Total

Per g

Total

Per g

9.6±0.6 ~1.4ti.3 8,6±0.8

b

SPENCER D. GREENDYKEet al. Table 5. Protein synthetic activity in golden hamsters exposed to control (14 light, l0 dark) and experimental (1 light, 23 dark) photoperiods. Week

4

12

Treatment

N

per mg

RNA

Control

5

3525 ± 691

19 +

4

Exper.

6

2922 ±

365

15 ±

3

31 +

1

Control

3

5368 ±

130

Exper.

3

5611 ±

1172

per

testes

(x 10 -6)

9 ± 4a

Values are expressed as mean _+ S.E.M. ~(P < 0.01}. experimental group (P < 0.05) only at 8 weeks. Moreover, no differences in the protein to DNA ratio were found within groups over time, however, a marked decrease (P < 0.01) was observed in experimental animals after 12 weeks of treatment. Table 5 shows the values obtained for cell-free protein synthesis expressed per unit of RNA and per testes. Protein synthesis was expressed per mg of RNA because RNA macromolecules participate in each step of protein synthesis, and the level of synthetic activity in the testicular cell-free system is directly proportional to the RNA content of the prep-, aration. The protein synthetic activity at 4 weeks of treatment did not differ between control and experimental groups, regardless of whether the data was expressed in dpm per mg of RNA, or dpm per testes. However, at 12 weeks of treatment a 73~o reduction in total dpm per testes was found to occur. This decrease was statistically significant (P < 0.01).

has been shown indirectly by Liu (1972), who reported that a 27~ decrease in testicular wet weight (following 12 weekks of treatment) resulted in a 149/o reduction in testicular cell numbers (a relative approximation of testicular DNA). The results from the present study show a 75Y,, decrease in mean testicular wet weight and a 34~ decrease in total testicular DNA; producing the same 2:1 ratio of wet weight loss to total DNA reduction, During the course of testicular regression, the amount of RNA per g of testes increased slightly in both ground squirrels and hamsters. However, the amount of protein per g of testes remained relatively constant, suggesting that these molecules are not being as actively produced or maintained. As would be expected, cellular DNA was well conserved and the amount of DNA per g of testes increased dramatically in both animals. Testicular cells seemed to decrease in size, as evidenced by the reduction in protein to DNA ratios over time. Cell-free protein synthesis by testicular PMS during DISCUSSION testicular regression remains constant when expressed Previous investigations with hamsters have demon- per mg total testicular RNA. However, when cell-free strated that shortened photoperiod brings about dis- testicular protein synthesis is expressed per testes, a tinct anatomical and histological involution of the dramatic decrease (73~o) in protein synthesis was testes (Hoffman & Reiter, 1965: Frehn & Liu, 1970; observed. Li & Goldberg (1976) have reported that Liu, 1972). Similar morphological results were in t,itro protein synthesis decreased during fasting in observed in the present study, namely a significant the extensor digitorum longus muscle from rats. Howreduction in testicular size and weight with season ever, when protein synthesis was expressed relative or shortened photoperiod. After 12 weeks of IL: 23D to the amount of RNA in the muscle, they found photoperiod, the weight of the testes decreased 76~/, no significant change in protein synthetic activity. in golden hamsters, and 3 months after emergence They proposed that the reduced protein synthesis from hibernation, the testes of 13-lined ground squir- observed in extensor digitorum longus was due to rels decreased 92°/,, However, this investigation marks a decrease in RNA content and not due to a change the first attempt to examine the effects of photoperiod in translational activity of the RNA. A similar on macromolecular parameters in the testes of phenomenon was observed during testicular regression. That is, the synthetic capacity of the various rodents. The total testicular content of RNA, DNA and pro- RNA species appears to be similar during testicular tein was found to decrease sharply in hamsters with regression because the protein synthetic activity per shortened photoperiod and in ground squirrels during mg RNA remains constant. Therefore, the decrease the brief active breeding season in the spring. in testicular protein synthesis appears to be related Although this decrease paralleled testicular regression to the decrease in the RNA content of the testes. If in both species, there was a much greater decrease this is true, the photoperiodically induced testicular in DNA content in the testes of ground squirrels than'" regression would appear to be transcriptionally conin the testes of hamsters. Specifically, at the comple- trolled. By regulating the amount of RNA produced. tion of the investigation, ground squirrels exhibited the level of protein synthesis in the testes during tesan 85~ reduction in testicular DNA content, while ticular regression could be controlled. To confirm this laboratory hamsters showed only a 34yo reduction hypothesis, it would be necessary to investigate the after 12 weeks of treatment. A decrease in testicular effect of short photoperiod on testicular RNA synDNA content of hamsters on a 8L: 16D photoperiod thesis.

Testicular RNA. DNA and protein content in hamsters Acknowledoemems--We express appreciation to Dr AI Katz for his assistance with statistical analyses, and to Terry Pease for his technical aide. This research was supported in part by Illinois State Research Grant 77-12, and by NIA Grant 1 R01 AG 00344-01.

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