Microphotometric measurement of some enzymatic activities in rabbit spermatozoa during epididymal maturation

Animal Reproduction Science, 13 (1987) 211-220

211

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Microphotometric Measurement of some Enzymatic Activities in Rabbit Spermatozoa during Epididymal Maturation B. FERRANDI, F. CREMONESI and F. PORCELLI

Institute of Anatomy of Domestic Animals, Faculty of Veterinary Medicine, University of Milan, Via Celoria, 10, 20133 Milan (Italy) (Accepted 17 September 1986)

ABSTRACT Ferrandi, B.: Cremonesi, F. and Porcelli, F., 1987. Microphotometric measurement of some enzymatic activities in rabbit spermatozoa during epididymal maturation. Anim. Reprod. Sci., 13: 211-220. Cytochemical quantitative measurements of isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH), cytochrome oxidase, lactate dehydrogenase (LDH), glucose 6-phosphate dehydrogenase (G6PDH) and glutamate dehydrogenase (GLDH) activities were made on rabbit spermatozoa collected from the testis, the different epididymal sites and the ductus deferens. These measurements were made on individual spermatozoa (at least 100 spermatozoa for each site under consideration) using a Vickers M 85 scanning microdensitometer. The activity patterns of the enzymes involved in the tricarboxylic acid cycle (ICDH, MDH) and in the respiratory chain (cytochrome oxidase) both showed a progressive decrease in the intramitochondrial oxidative metabolism from the testis to the ductus deferens. This was in contrast to LDH activity which represents the anaerobic glycolysis pathway rather than the activity of intramitochondrial LDH. The G6PDH activity could be related to those membrane modifications which the male gamete undergoes during its epididymal maturation. Potential GLDH activity was relatively intense in the spermatozoa from the testis and from the initial and distal segments of the genital tract, suggesting an intramitochondrial synthesis of enzymes such as cytochrome oxidase or ATPase. The quantitative variations of the enzymatic activities occurring during the transit of spermatozoa along the male genital tract suggested the existence of different specific interactions between the spermatozoon and the epididymal microenvironment.

INTRODUCTION T h e E u t h e r i a n m a m m a l i a n s p e r m a t o z o o n u n d e r g o e s a series of p r o f o u n d m o r p h o l o g i c a l a n d p h y s i o l o g i c a l m o d i f i c a t i o n s , b o t h in its p a s s a g e a l o n g t h e m a l e genital t r a c t , w h e r e it r e a c h e s its f u n c t i o n a l m a t u r i t y , as well as in t h e female g e n i t a l t r a c t d u r i n g c a p a c i t a t i o n ( B e d f o r d , 1970, 1975; C h a n g a n d

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Hunter, 1975; Austin, 1985). The achievement of functional maturity in the spermatozoon is related to many modifications in its metabolic activity (for review see: Voglmayr, 1975; Austin, 1985). The biochemical investigations carried out in vitro on spermatozoa metabolism represent only the capacity of the sperm under the given incubation conditions and do not necessarily reflect the metabolism of spermatoza in vivo (Inskeep and Hammerstedt, 1982). Nevertheless, the interaction in vivo between the spermatozoon and its environment is deemed particularly important in that the spermatozoon's behaviour in vivo could be greatly conditioned by the particular composition of the genital fluids ( Orgebin-Crist et al., 1975; Voglmayr, 1975; Austin, 1985). In the present study we have attempted to integrate methods of biochemical analysis with those of cytochemical investigation. Some of the limitations of the biochemical approach (i.e. the obtainment of findings which represent, at best, mean values and the need for large quantities of sufficiently homogeneous cells) can be avoided by cytochemical methods which require far fewer cells. Cytochemical measurerSents require single cellular elements and provide information on the variation in metabolic behaviour. In the present study, cytochemical quantitative determinations on isocitrate dehydrogenase (ICDH), malate dehydrogenase ( M D H ) , cytochrome oxidase, lactate dehydrogenase (LDH), glucose 6-phosphate dehydrogenase (G6PDH) and glutamate dehydrogenase (GLDH) activities were carried out using rabbit spermatozoa present in the testis and along the various sections of the epididymis. MATERIALSAND METHODS Investigations were carried out using spermatozoa from eighteen 8-monthold New Zealand White male rabbits. The animals were first killed by cervical dislocation and then spermatozoa collected from the testis, epididymis (proximal and distal caput, corpus, proximal and distal cauda, according to Moore, 1980) and the pelvic portion of the ductus deferens. Testicular imprints and smears of spermatozoa were air-dried for 3 h and then processed according to cytochemical procedures for detecting the following enzymatic activities: (a) ICDH, (b) MDH, (c) LDH, (d) G6PDH, (e) GLDH and (f) cytochrome oxidase. The reactions (a), (b), (c), (d) and (e) were carried out according to methods reported by Pearse (1961). The reaction (f) was carried out according to Frasch et al. (1978). Each histoenzymologic reaction was based on either tetrazolium salts reduction (a,b,c,d,e) or on the oxidative polymerization of diaminobenzidine (f) and can be applied to a quantitative cytochemical assay (Cabrini et al., 1969; Frasch et al., 1978; Henderson and Loveridge, 1981 ). Microdensitometric measurements (expressed in arbitrary units of inte-

213

grated optical density) were made using a Vickers M85 integrator microdensitometer at wavelength ~ = 585 + 5 nm for formazan obtained from NBT (pnitro tetrazolium blue, Sigma Chemical Company, St. Louis, U.S.A.) reduction, and at ~ = 480 + 5 nm for the final reaction product obtained from oxidation of 3,3'-diaminobenzidine tetrahydrochloride (BDH Chemicals Ltd., Poole, England). The microdensitometer was used under the following conditions: 100 X oil immersion objective, 10 X eyepiece, dry condenser and 0.4/zm diameter flying spot. The cytophotometric errors due to glare and non-specific light loss under such conditions, and evaluated according to Bedi and Goldstein (1974), were found to be negligible ( < 3%). Consequently no instrument correction was introduced. At least 100 measurements were taken per animal for each cell population for a total of 2100 measurements for each cytochemical technique. The differences of integrated optical density values in spermatozoa of different populations were statistically evaluated by Student's "t" test. Only results which differed by P < 0.01 were regarded as statistically significant. RESULTS All the measured enzymatic activities were detectable cytochemically in the mid-piece of the spermatozoon. The integrated optical density values are reported in Table 1 and shown diagrammatically in Figs. 1-6. The integrated optical density values of ICDH, MDH and cytochrome oxidase activities (Figs. 1, 2, 3) gradually declined (P < 0.01 ) from the testis to the proximal and distal caput and corpus epididymis, increased ( P < 0.01 ) in the epididymal proximal cauda, finally decreasing ( P < 0.01 ) again in the epididymal distal cauda and in the ductus deferens. The LDH activity (Fig. 4) increased significantly from the testis to epididymal proximal caput, showed some fluctuations in the epididymal distal caput and in the epididymal corpus, significantly decreased in the epididymal proximal cauda, significantly increased in the epididymal distal cauda and then significantly decreased in the ductus deferens. The G6PDH activity (Fig. 5 ) decreased significantly from the testis to epididymal distal cauda. The values then increased significantly in the ductus deferens, though remaining significantly lower than those of the testicular spermatozoa. The GLDH activity (Fig. 6) increased (P<0.01) from the testis to epididymal proximal caput; decreased ( P < 0.01 ) in both the distal caput and in the epididymal corpus and then increased gradually and significantly, reaching levels similar to those of the testicular spermatozoa, in the ductus deferens.

3.86 ± 0.53 "b 4.35+0.50 ~ 3.83 ± 0.47 "b 6.42+1.50 a 5.73-----1.07a 8.68+1.18 a 2.93 ± 0.46 ab 3.90+0.70 a 4.07 ± 0.60 a

10 11 12

13 14 15

16 17 18

3.29 ± 0.49 c 4.38±0.62 b 4.48 ± 0.42 b

3.09±0-33 b 4.48i0.55 b 5.71+0.825

4.18 ± 0.59 ~ 4.94--+0.50 b 4.75 ± 0.65 ¢

2.54+0.50 bc 2.05+0.65 b 2.49+0.60 b

3.51+_0.83 ~ 3.00+0.64 ~ 3.94+1.14 ~

7 8 9

5.65+1.44 b 3.98 ± 0.66 b 3.30+0.83 b

6.84+3.13 a 6.13 ± 0.90 a 5.19+1.08 ~

4 5 6

2.48-+0.55 b 2.94+0.70 b 2.79--+0.52 b

proximal caput

Epididymis

5.10+1.19 ~3 4.16+1.19 ~ 4.72+1.07 ~

Testis

Spermatozoa localizations

1 2 3

Animals

2.54 ± 0.70 d 3-18+0-27 c 3.41 ± 0.48 c

1-89 ±0. 46c 4.10±0.685 4.13+0.35 ~

3.96 ± 0.42 "¢ 4.73+0.65 b 4.38 ± 0.61 d

1.81±0.52 d 1.70+0.60 ~ 1.62+0.57 ¢

4.91± 1.33 c 3.68 ± 0.58 b~ 2.85±0 .52°

2.20+0.39 b 2.14--+0.59 ¢ 2.26+0 "39¢

distal caput

1.47 ± 0.33 e 2.67±0.28d 2.53 ± 0.35 d

1.55i0.20c 3 "64±0"72c 3"71 +0"34d

3.76 ± 0.54" 4.97±0"63b 4.24 ± 0.55 d

0.94_+0.79 ~ 1.02_+0.54 d 0.79_+0.41 d

2.32±0.89d 2.98 ± 0.63 d 1"08+0"660

1.29 ± 0.43¢d 1"35+--0"640 1'76+0"340

corpus

2.70 i 0.37 ad 3.03i0"35c 3.65 ± 0.29 Ce

1"05±0"24d 1"24±0"28d 2"39±0"26e

3.37 ± 0.40 c 3"32 ±0"40c 3.75 ± 0.48"

2.80±0.585 2.21+0.79 b 3.32+0.52 ~

4"17±0"96e 4.66 ± 0.68 ¢ 3"48±0"50¢

2.21 ±0"45b 1"37±0"45~ 2"27±0"41~

proximal cauda

3.12 ± 0.42 bc 3"66±0"22a 3.94 ± 0.51 ~e

0"65i0"20e 0"74±0"23~ 1"42±0"32f

4.06 ± 0.57 bc 3"98±0"37d 4.12 ± 0.45 bd

2.23+_0.56 ¢ 1.68±0.59 c 2.76+0.56 b

2"72 ±0"76d 3.59 ± 0.45 ¢ 2"56±0"48b

1"56±0"41¢ 2"23 ±0"58¢ 1"58±0"25d

distal cauda

3.83 ± 0.31 f 3"97±0'34a 4.01 ± 0.43 ae

1"04i0"21d 2"15±0"17f 2"53±0"35~

2.89 ± 0.24 d 3"05±0"28° 3.28 ± 0.37 ~

1.50_+0.62 d 1.00_+0.44 d 1.71±0.45 °

1"50±0"58f 2.47 ± 0.62 f 1"64±0"62d

1"15+0"470 1"07 ± 0"48dc 1"15±0"34~

Ductus deferens

1Cytochemical microdensitometric determinations carried out as indicated in Materials and Methods. All values of integrated optical density are expressed in arbitrary units as mean _+S.D. of determinations carried out on at least 100 spermatozoa. 2ICDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; LDH, lactate dehydrogenase; G-6PDH, glucose-6 phosphate dehydrogenase; GLDH, glutamate dehrydrogenase.

GLDH

G-6PDH

LDH

Cytochrome oxidase

MDH

ICDH 2

Enzymatic activities

Enzymatic activities in rabbit spermatozoa collected from the testis and different sites of the male genital tract I

TABLE 1

215 MDH

ICDH

8~ 76-

_E

5~ 4:

~6

23

d4

~4

m

~-sg~

(3

3-

(i)'3 a

6. b

c

d

e

f

g

5

Male genital tract different sites 4

Male g e n i t a l t r a c t d i f f e r e n t s i t e s

Fig. 1

Fig. 2.

Fig. 1-6. Isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH), cytochrome oxidase, lactate dehydrogenase (LDH), glucose 6-phosphate dehydrogenase (G6PDH) and glutamate dehydrogenase (GLDH) activities in rabbit spermatozoa collected from the testis and different sites of the male genital tract (a = testis; b = proximal caput, c = distal caput epididymis; d = corpus epididymis; e--proximal cauda, f = distal cauda epididymis; g = ductus deferens - - (see Table 1 ). Vertical lines represent the standard deviation of the mean. DISCUSSION

The results obtained for ICDH, MDH and cytochrome oxidase activities characterize changes in the oxidative metabolism in rabbit spermatozoa. The

216 CYTOCHROME OXIDASE

LDH

22 0'1o

v t

~3

0"11 nc~

0"12 T a

b

c

d

e

f

g

Male genital t r a c t d i f f e r e n t s i t e s Fig. 3.

Male g e n i t a l t r a c t d i f f e r e n t

sites

Fig. 4.

general trends in these enzymatic activities were similar and showed a general decrease in the spermatozoal intramitochondrial oxidative metabolism from the testis to the ductus deferens. Our data contrast with biochemical findings reported by other authors that tend to attribute to the spermatozoa of other species (i.e. ram, bull, boar, rat, guinea pig) a constant respiratory activity during epididymal transit or an increase in such activity in the terminal parts of the male genital tracts (Voglmayr et al., 1967; Frenkel et al., 1973; Voglmayr, 1975; Paz et al., 1978; Dacheux and Paquignon, 1980; Hammerstedt, 1981; Inskeep and Hammerstedt, 1982). The three enzymatic activities involved in the intramitochondrial oxidative metabolism increased in spermatozoa in the proximal cauda. Such a variation could be considered the consequence of a generalized increase in the metabolic activity of the spermatozoa in this site, even if this acceleration of the overall metabolic activity might not concern all the various metabolic pathways in equal measure, as suggested by Inskeep and Hammerstedt (1982). Glycolysis and respiration constitute the main sources of energy for motility in mammalian spermatozoa and the variations observed could depend on the great variation of exogenous (hexoses, lactate, pyruvate, intermediate products of the tricarboxylic acid cycle and fatty acid) or endogenous ( lipids ) sub-

217 G~PDH

41

~6 co v 4

GLDH

g2 4~

5

~ v

4

b3

i,

(~17

o 5

~

dis Male genital tract different sites

Fig. 5.

2.

dis a

b

c

d

e

f

Male genital tract different sites

Fig. 6.

strates available for use by the spermatozoon in its energetic metabolism (Scott et al., 1967; Evans and Setchell, 1979, a,b). The metabolic behaviour of the spermatozoa may depend on the interactions with epididymal fluids which vary in composition at the different duct sites. The epididymal environment would exert an essential role in the metabolism and development of the male gamete ( Setchell and Hinton, 1981; Austin, 1985). The LDH activity pattern supports the hypothesis that a general decrease in the anaerobic glycolysis from the testis to the ductus deferens occurs in rabbit spermatozoa during epididymal transit. About 2-6% of the total lactate

218 dehydrogenase activity cytochemically detectable is ascribable to an intramitochondrial LDH-X isoenzyme which enables lactate to be utilized directly as a substrate for oxidative phosphorylation ( Storey and Kayne, 1977; Voglmayr et al., 1977). Some of our data are not in agreement with the results obtained in other species where a considerable increase in the anaerobic glycolysis has been demonstrated in the spermatozoa of the epididymis or of the ejaculate as compared with the anaerobic activity of the spermatozoa present in the testis. These conclusions were reached on the basis of the quantity of glucose converted to lactic acid in vitro, or of the conversion rate of glucose to lactic acid and of the magnitude of the Pasteur effect (Voglmayr et al., 1967; Frenkel et al., 1973; Voglmayr, 1975; Voglmayr and White, 1979; Hammerstedt, 1981; Inskeep and Hammerstedt, 1982 ). The G6PDH activity indicated the presence of a pentose shunt in the rabbit spermatozoa along the genital tract. This pathway is involved in lipogenic activity in testicular spermatozoa while during epididymal transit it could be related to sperm membrane modifications. The biochemical modifications in membrane composition are further underscored by the qualitative and quantitative variations in the composition and i n t h e degree of saturation of fatty acids (Evans and Setchell, 1979a,b). The very high G6PDH activity of testicular spermatozoa could also suggest that in the rabbit, as in the buffalo, the pentose phosphate cycle could be involved in energy production ( Sidhu and Guraya, 1979 ). In the epididymal cauda and in the ductus deferens the increase in GLDH activity could be related to processes of intramitochondrial protein synthesis without nuclear transcriptional activity. This activity has been hypothesized by Bragg and Handel (1979) for mouse spermatozoa present in the epididymal cauda and in the ductus deferens; the products of such a synthesis are probably enzymes - - a cytochrome oxidase and an ATPase - - involved in the production of energy for the spermatozoa motility. An intramitochondrial protein synthesis may allow sperm to utilize new enzymes or isoenzymes in its transit through the male and female genital tracts (Bragg and Handel, 1979). In conclusion, our data suggest the existence of a definite interaction between spermatozoon and microenvironment and allow a more precise description of the modifications of the metabolic activity of the spermatozoon in its passage along the male genital tract, as compared with the results obtained by means of biochemical research alone. We think it is particularly important to emphasize the decrease of all the enzymatic activities studied, with the exception of that of LDH, in the epididymal corpus. Equally significant are the high levels of enzymatic activity found in the spermatozoa in the epididymal proximal cauda and of LDH activity in the epididymal distal cauda, as well as a general decrease in the metabolic activities in the spermatozoa of the ductus deferens.

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