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International Journal of Biological & Medical Research

Int J Biol Med Res. 2012; 3(4): 2560-2564

“Effect of salivaryadenectomy of the pregnant mother on testicular lactase

dehydrogenase in mice”.

Bodare R. D., Pillai M. M. Department of Zoology , Shivaji University, Kolhapur – 416004 (M.S.)

A R T I C L E I N F O A B S T R A C T

Keywords:

Original Article

Tuberculous lymphadenopathyFNACImmunochromatographic test(ICT-TB)

The large body of evidences indicate that epidermal growth factor (EGF) is synthesized, stored

and secreted by the submandibular glands and also by the sublingual glands. EGF shows

stimulating activity on various epidermal epithelial tissues both in vitro and in vivo. A series of

observations suggest that the growth factors and in particular EGF plays an important role in

the male reproductive system. The concentration of EGF in the submandibular gland and

plasma of the female mice increases substantially during pregnancy. The midgestational

sialoadenectomy of female mice reduces EGF level. To show the effect of salivariadenectomy of

the pregnant mother, on the development and function of testis, testicular lactase

dehydrogenase isoenzyme was studied biochemically and electrophoretically. LDH-X is one of

the LDH isoenzymes which is unique to pachytene spermatocytes, spermatids and mature

sperm. The pregnant females were salivariadenectomised (sublingualectomy,

sialoadenectomy and both sublingualectomy + sialoadenectomy) at 10th day of their gestation

period. The 20 days, 45 days, 60 days old male offsprings of salivaridenectomised mother as

well as male offsprings of the same age of sham operated mother were sacrificed by the cervical

dislocation and their testis were used to study the electrophoretic separation of lactate

dehydrogenase and estimation of lactate dehydrogenase. In the testis of 20 days, 45 days, 60

days normal mice of sham operated mother, the LDH was separated into six bands i.e. I, II, III, IV,

V and LDH-X. But in the testis of above aged mice of sublingualctomised, sialoadenectomised

and salivariadenectomised mother, LDH-X band was disappeared. The LDH activity was

decreased significantly in the testis of 20 days, 45 days, 60 days offsprings of

sublingualectomised mother as compared to normal mice of sham operated mother. The above

results suggest that maternal EGF affects the development and functions of testis.

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1. Introduction

The submandibular gland of mice is rich source of epidermal

growth factor (EGF) (Barka, 1980). A large body of evidences

indicate that EGF is synthesized, stored and secreted by the cells of

granular convoluted tubular cells of submandibular glands and also

by the cells of sublingual glands. Epidermal growth factor is single

polypeptide having aspargine at NH2 – terminus and arginine at

COOH- terminus and consists of 53 amino acids having molecular

weight about 6,000 daltons (Taylor et. al 1970). EGF is known to

elicit a wide variety of cellular responses including membrane

changes, activation of various transport systems and changes in

cellular metabolism in addition to stimulating mitogenesis. All

these are brought about by the action of EGF upon its specific cell

surface receptors (Staros et. al., 1985).

EGF was first detected in submandibular gland around 20th

postnatal day in GCT cells of male mice and 30th day of age in

female mice (Gresik and Barka, 1978, Barka, 1980). The role of EGF

in the control of spermatogenesis has been the subject of intense

investigation and several observations suggests that growth

factors and in particular EGF play an important in the male

reproductive system (Kurachi and Oka, 1984., Richards et. al.

1984., D'Cruz et. al. 1989; Pillai and Walvekar, 2002). EGF has been

Copyright 2010 BioMedSciDirect Publications IJBMR - All rights reserved.ISSN: 0976:6685.c

* Corresponding Author : Dr Geetha J.P

Assistant Professor, Department of pathology.Sree Siddhartha Medical college, Tumkur.Phone No: 9342972556. Email: [email protected]

Copyright 2010 BioMedSciDirect Publications. All rights reserved.c

Bodare R. D & Pillai M. M. Int J Biol Med Res. 2012; 3(4): 2560-2564

shown to be increased in the submandibular glands and plasma

during pregnancy in mice and thus is one of the potential regulators

of the foetal development (Tsusumi and Oka, 1987).

Mammalian spermatogenesis is complex process of cellular

differentiation involving unique cytological and molecular

processes that culminate in the formation of spermatozoa. So to see

the effectiveness of spermatogenesis y emphasis was given to

enzymes and proteins that appear during spermatogenesis. This

was best seen with lactate dehydrogenase (Xue and Goldberg, 2000).

Lactate dehydrogenases (L-Lactate, NAD- oxidoreductase,

EC1.1.1.27; LDH) are glycolytic isoenzymes that catalyse

interconversion of lactate and pyruvate with NAD+ as coenzyme.

The structure of lactate dehydrogenase has been investigated by a

number of investigators (Cahn et. al. 1962, Everse and Kaplan 1973,

Li et. al. 1989), who have shown that LDHs are tetramere composed

of two subunits as 'A' ('M' or muscle or type -5) and 'B' ('H' or Heart or

type-1). The two subunits 'A'/M and 'B'/'H' combine to form two

pure types of isoenzymes H4/A4(LDH-1), M4/B4(LDH-5) and three

hybrid isoenzymes as H3M/A3B (LDH-2), A2B2/H2M2(LDH-3),

HM3/AB3 (LDH-4). These two subunits A and B are under separate

genetic control, encoded by Ldha gene and Ldhb gene. LDH-A

isoenzyme is better known for pyruvate reduction in anerobic tissue

(muscle) whereas LDH-B isoenzyme is better for L-Lactate oxidation

in aerobic tissue (Heart and Brain), (Holbrook et. al. 1975, Markert

et. al. 1975).

Testis contain above five different isoenzymes of Lactate

dehydrogenase as LDH-1, LDH-2, LDH-3, LDH-4, LDH-5. In addition,

testes contain sperm specific unique isoenzyme known as LDH-X.

LDH-X isoenzyme is encoded by separate gene loci Ldhc gene. The

LDH-X isoenzyme is found in the testis, spermatocytes, spermatids

and sperms (Zhinkham et. al. 1964, Stanbaugh and Buckley, 1967).

Lactate dehydrogenase LDH-X is adapted to satisfy the

metabolic requirements of the differentiating germ cells and

functional spermatozoa (Blanco, 1980). LDH-X is one of the best

characterized germ cell specific isoenzyme that plays important role

in the process of spermatogenesis. The testis specific mouse lactate

dehydrogenase c (mldhc) gene is transcribed at high levels, in

germinal epithelial cells. LDH-X protein appears at pachytene,

becomes more abundant in round and elongating spermatids, and is

present in mature spermatozoa. Cloning, sequencing and analysis of

the mldhc promoter reveled a 100-bp core region that was defined

by its ability to direct testis specific expression of reporter gene in

vitro transcription assays (Zhou et. al. 1994).

LDH-X is differentiated in catalytic, enzymatic and

immunologically from LDH-1 and LDH-5. The amino acids, histidine,

glutamic acid, praline, alanine, valine, isoleucine, leucine and

tyrosine vary only slightly or not at all between LDH-1 and LDH-5

(Weiland et. al. 1961, Pesce et. al. 1967). In LDH-X, isoleucine is low

and leucine is exceptionally high to that of other Lactate

dehydrogenase isoenzymes. Similarly glycine and threonine

content of LDH-X is also much higher than other isoenzymes. The

differences in primary structure of LDH-X, with the previously

reported immunochemical (Goldberg, 1971) and genetic (Blanco,

et. al. 1964) data, clearly established that LDH-X is distinct than

epigenetic modification of A or B subunits or both.

One of the catalytic properties distinguishing its broader

substrate specificity and high affinity for lactate than other LDH –

isoenzymes (Goldberg, 1977, Wheat and Goldberg, 1983). LDH-X

from mouse and rat, for example is able to catalyses the NAD+

linked interconversion of 2-oxoacid –2hydroxyacid with a variety

of substrates including 5 and 6- carbon branched chain

compounds like (4-methyl-2-oxopentanoate, 3-methyl-2-

oxoacids) are the products of transamination reactions with

amino acid leucine, isoleucine and valine respectively.

Furthermore, LDH-X is more sensitive to lactate inhibition than the

other somatic type LDH isoenzymes (Goldberg, 1977; Mita and

Hall, 1982; Nakamura et. al. 1984).

As far as immunologic functions are concerned the LDH-X from

sperm and somatic LDH-1 and LDH-5 share functionally related

antigenic epitopes that can generate immune response in vivo and

in vitro with qualitative differences. However, immunosuppressive

determitant of LDH-X is well specific and dose selective (Gupta and

Chatuevedi, 2000). LDH-X has been successfully used as an antigen

to prvoke antibodies interact with sperm in the female

reproductive tract and effectively block fertilization. (Goldberg,

2000).

Whether the role of LDH-X is strictly metabolic (i.e. glucolysis)

or is also immunological, LDH-X undoubtedly plays significant part

in sperm physiology and this must be reflected in its unique

properties.

M a t e r i a l : - M a l e m i c e ( M u s m u s c u l u s ) o f

salivariadenectomised mother and sham operated mother were

used for the investigation. Breeding pairs were obtained from

Hindustan Antibiotics Ltd. Pune. They were reared in

departmental animal house having air conditioned rooms. They

were maintained in cages of dimensions (10” x 8” x 5”) and allowed

to live in 3 to 4 numbers. They were supplied with Amrut mice feed

(Pranav Agro Industries) and clean (aquaguard) water ad libitum.

For this study, pregnant female mice were selected. The

pregnant females of three months old were weighed and

salivariadenectomised at the 10th day of gestation period.

Salivariadenectomization process includes removal of salivary

glands i.e. sublingual glands, and submandibular glands alone and

together. Thus sublingualectomy means removal of sublingual

glands, sialoadenectomy is removal of submandibular glands and

salivariadenectomy includes removal of both sublingual and

submandibular glands.

2. Material And Methods:

2561

1) Polyacrylamide Gel Electrophoresis of Lactate

Dehydrogenase.(Ornstein, 1964; Davis, 1964; Andrews, 1981)

Polyacrylamide gel electrophoresis (PAGE) was performed in

the cylindrical glass tubes. The glass tubes used for gel preparation

were having 5 mm diameter and 8 cm in length.

Gel Preparetion: 5% PAG:Acryl amide -4.75 gm, Bis acryl

amide-0.25 gm, TEMED-0.05 gm (N,N,N',N' tetra methyl ethylene

diamine) Ammonium per sulfate- 0.001%, Prepared in 100 ml

separation gel buffer pH 7.5.

Separation Gel Buffer pH- 7.5

Tris (Hydroxymethyl) – 0.856gm, 1MHCl – 6ml; dissolved in 100

ml distilled water.

Electrode chamber buffer:

At Anode- 0.26 M Tris buffer pH (9.1), Tris (Hydroxymethyl-

Aminoethane)-25.2 gm, EDTA (Ethylenediamine tetra acetic acid)-

2.5 gm, Boric acid-1.9 gm, dissolved in 100ml distilled water.

At Cathode-Barbital buffer (pH 8.6)- Sodium diethyl barbiturate

-5.12 gm, Diethyl barbituric acid, 0.92 gm dissolved in 1000 ml

distilled water.

Preparation of Sample: Sample was prepared by

homogenization of testis (25mg/ml distilled water) in refrigerated

morter and pestle. Homogenized tissue was centrifuged at 100C

with 5000 rpm for 10 minutes. The supernatant was used as sample.

Sample dye: 1ml supernatant + 50 mg sucrose + 0.3 ml glycerol + 1

mg Bromophenol blue. The protein concentration in the sample was

estimated by the method of Lowry et. al. (1951). The sample

containing 40µg protein was loaded for single gel rod with the help

of microsyringe.

Electric supply: The voltage was kept constant at 150 volt during

the operation of electrophoresis and 3mA current per rod was

employed.

Gel staining:

A) Staining mixture- 1) 1M buffer substrate (Lactate): 1ml of 60%

sodium lactate was diluted to 10ml with M/15 phosphate buffer pH

7.5.( M/15 phosphate buffer – 84.1 parts of Na2HPO4 + 15.9 parts

KH2PO4 to 100ml distilled water).

1) Nitroblue tetrazolium chloride (N.B.T.)- 1mg/ml distilled water.

2) Phenazine methosulphate (P.M.S.)- 1mg/ml distilled water.

B) Working solution: 1ml buffered substrate (Lactate); 3ml N.B.T.,

0.14 ml P.M.S., 1 ml LDH buffer (M/15 phosphate buffer pH 7.5); 10

mg NAD+.

The gels were removed from gel tube by microsyringe in

petridish containing cold distilled water. The gels were then put in

container with working solution. Incubated at 370 c until bands

were resolved.

2) Estimation of Lactate Dehydrogenase- (Sevela and Tovoek,

1989): The method is based upon the measurement of the rate of

pyruvate formation during lactate oxidation reaction is assisted by

lactate dehydrogenase of the sample.

For estimation of LDH the sample was prepared by

homogenization of testis (1mg/1ml distilled water) which is then

centrifuged at 100C with 5000 rpm for 10 minutes. The

supernatant was used as sample.

The procedure for the estimation of LDH was carried out

according to method of Sevela and Tovorki (1981). Measurement

of absorbance was taken for sample solution on the Spectronic 20

colorimeter at 560 nm. The activity of enzyme was estimated by

making use of an analytical curve.

The testicular lactate dehydrogenase was separated

electrophoretically into six bands i.e. I, II, III, IV, V and LDH-X. The

LDH-X band was observed very close to origin. (Fig.1)

In the male offsprings of the same ages of sublingualectomised,

sialoadenectomised and salivariadenectomised mother, the LDH

was separated electrophoretically into five bands i.e. I, II, III, IV, V.

The LDH-X band was completely disappeared from the testis of

above offsprings. In addition other changes were also observed.

In 20 days male offsprings of sublingualectomised,

sialoadenectomised and salivariadenectomised mother were

separated having same staining intensity, which was very low

compare to normal (LDH-1 to LDH-5). Similarly there were also

changes in LDH-1 to LDH-5 bands in offsprings of 45 days and 60

days of sublingualectomised mother. But in the offsprings of 45

days of sialoadenectomised mother, LDH-3 and LDH-4 stained

faintly. In 90 days old offsprings of sialoadenectomised mother

band no. 5 absent while in salivariadenectomised case, LDH band

no. 4,5 absent.

Again in the 45 days offsprings of salivariadenectomised

mother, LDH-3, LDH-4, LDH-5 bands stained faintly. In 60 days

offsprings of sialoadenectomised mother, LDH-4 band was

disappeared and in the offsprings of salivariadenectomised

mother, the LDH-3, LDH-4 bands stained very faintly.

LDH activity in m mol h-1 L-1 min per mg protein was

estimated from the testis using lactate as a substrate and NAD+ as a

coenzyme. In sham operated mothers, male offsprings of 20 days

the testicular LDH activity was 2.23 + 0.6330. B u t t h e L D H

activity was decreased not significantly ( ) in the testis of

offsprings of a sublingualectomised mother ( ) but significantly

decreased in sialoadenectomised ( ) and salivariadenectomised

mother. (Table No.1).

3. Results


2562

Animals

Animals

Animals

Age in days

Age in days

Age in days

LDH concentration

LDH concentration

LDH concentration

't' value

't' value

't' value

Statistical significance



Control

Sublingualectomised

Mother's male

Sialoadenectomised

Mother's male

Salivariadenectomised

Mother's male

Control

Sublingualectomised

Mother's male

Sialoadenectomised

Mother's male


Mother's male

Control

Sublingualectomised

Mother's male

Sialoadenectomised

Mother's male


Mother's male

20

20

20

20

45

45

45

45

60

60

60

60

2.23 ± 0.6330

1.80 0.7219

1.74 0.3968

1.37 0.5645

±

±

±

3.48 0.3286

2.88 0.3286

2.52 0.4724

2.14 0.5459

±

±

±

±

4.44 0.4242

3.84 0.4242

3.24 0.4242

3.00 0.4320

±

±

±

±

1.138

2.3191

4.1314

4.5648

5.8980

7.4345

3.5360

7.0721

9.4886

P< 0.05

P< 0.001

P< 0.001

P< 0.01

P< 0.001

P< 0.001

P< 0.02

P< 0.001

P< 0.001

Table No.1. Effect of salivariadenectomy of pregnant mother

on LDH activity in testis of male offsprings (20 days).





-1 -1(LDH activity in m mol h L min per mg protein); values are mean

+ S.D.


+ S.D.


+ S.D.

The LDH activity in the testis of 45 days mice of sham operated

mother was 3.48 + 0.3286. but this LDH activity was decreased

significantly (P< 0.01) in the offsprings of sublingualectomised

mother, the LDH activity decrease was highly significant (P< 0.001)

in the offsprings of sialoadenectomised mother and further more

decrease in LDH activity was observed in the offsprings of

salivariadenectomised mother (P< 0.001). (Table No.2)

The LDH activity in the testis of 60 days mice of sham operated

mother was 4.44 + 0.4242. There was decrease in the LDH activity

in the offsprings of the sublingualectomised mother as compared

to normal and this decrease was significant (P< 0.01). But in the

offsprings of the sialoadenectomised mother the decrease in LDH

activity was highly significant (P< 0.001) and again it gets

decreased further more in the offsprings of salivariadenectomised

mother. This decrease was also highly significant (P< 0.001).

In 90 days old mice of sham operated mother, the LDH activity

was 5.24 + 0.4242. The enzyme activity decreased in the testis of

sublingualectomised was decreased but in testis of offsprings of

sialoadenectomidsed and salivariadenectomised mother the

enzyme activity was highly decreased (P< 0.001).


2563

4. Bibliography

19) Pillai, M.M. and Walvekar, M.V. (2007) : Sublingualectomy effect on testis and spermatogenesis. Diversity and Life processes from Ocean and Land. Editors-P.V. Desai and R.Roy ,Goa University : 114-123.

20) Reys, A.B.V. and Wakasugi, N. (1995). Long term influence of sialoadenectomy on reproductive performance of male mice. J. Reprod. Fertil. 105:279-285.

21) Russel, L.D.; Weis,T.; Goh, J.C.; Curl, J.L. and Liu, A.L.(1990). The effect of submandibular gland removal on testicular and epididymal parameters. Tissue Cell, 22: 263.

22) Sevella and Tovarek. (1989). “Lactate Dehydrogenase”, Laboratory manual in Biochemistry. Ed.by: Stoev, E.A. and Mrovaka, V.G.; MIR Publishers, Moscow,106.

23) Skude, G.,Von, Eyben, F.E. and Kristeiansen, P. (1984). Additional lactate dehydrogenase (LDH) isoenzyme in normal testis and spetrmatozoa of adult man. Mol. Gen. Genet. 198(1): 172-174.

24) Stubbs, S. C., Hargreave, T. B., Habib, F. K. (1990): Localization and characterization of epidermal growth factor receptors on human testicular tissue by biochemical and immunohistochemical techniques. J. Endocrinol. 152: 485-492.

25) Suarez. Quian, C.A and Niklinski,W. (1990) :Immunocytochemical localization of the epidermal growth factor receptor in mouse testis. Biol.Reprod. 43:1087-1097.

26) Taylor, J.M.; Cohen, S. and Mitchel, W.M. (1970). Epidermal growth factor, high and low molecular weight forms. Proc. Natl. Acad.Sci.USA. 67 :164.

27) Tsutsumi, T.; Kurachi, H. and Oka, T. (1986). A physiological role of epidermal growth factor in male reproductive function. Science, 233 : 975.

28) Virji, N. (1985): LDH C4 in human seminal plasma and its relationship to testicular function, Methodological Aspects. Int. J. Androl. 8: 193-200.

29) Wheat, T.E. and Goldberg, E. (1983). Sperm specific lactate dehydrogenase, C4: antigenic structure and immunosuppression of fertility. Isozymes: Curr.Topics Biol.Med. Res. 7 : 113-130.

30) Zhinkham, W.H.; Blanco, A. Clowry, J. (1964). An unusual isoenzyme of lactate dehydrogenase in mature testis: localization, ontogeny and kinetic properties. Ann. N.Y.

1) Andrew, A. T. (1981). Electrophoresis; theory, technique and biochemical and clinical applications. Clarendon Press, Oxford, 50.

2) Barka, T. (1980). Biologically active polypeptides in submandibular glands J.Histochem. Cytochem. 28: 836-869

3) Bodare, R.D. and Pillai, M.M. (2007) : Effect of saliadenectomy on the testicular lactate dehydrogenase –X in mice. J. Cell Tissue Res, 7(2) : 1083 -88.

4) Cahn, R.D.; Kaplan,N.O.;Levine,L.; willing, E. (1962). Nature and development of lactic dehydrogenases. Science.136:962.

5) Davis, B.J.(1964). Disc electrophoresis II Method and application to human serum proteins, Ann.N.Y.Acad.Sci.121: 404.

6) Gavella, M. and Cvitkovic, P. and Skrabalo, Z. (1982). Seminal isoenzyme LDH-X in infertile men. Andrologia. 14 : 104 – 109.

7) Gerez de Burgos., N.M. Burgos, C. Coronel, C.E.., (1979): Correlation of Lactate dehydrogenase isoenzyme C4 activity with the count and motility of spermatozoa. J. Repro. Fertil. 55: 107-111.

8) Goldberg, E.(1971). Immunocytochemical specificity of lactate dehydrogenase-X, Proc. Natl. Acad. Sci.USA. 68 : 349-352.

9) Goldberg, E. (1977). Isozymesin testis and spermatozoa. Isoenzymes; Curr. Topics Biol. Med. Res. 1 :79-124.

10) Gray, A. ; Dull, T. J. and Ullrich, A.: Nucleotide sequence of epidermal growth factor cDNA predicts a 1,28,000 molecular weight protein precursor. Nature.303: 722 725. (1983).

11) Gupta, C. (1996). The role of Epidermal Growth Factor Receptor (EGFR) in male reproductive tract differentiation: stimulation of EGFR expression and inhibition of Wolffian duct differentiation with anti-EGFR antibody. Endocrinol. 137(3): 905-910.

12) Lowry, C.H.; Rosebrough, N.J.; Farr, A.L.; Ranall, R.J. (1951). Protein measurement with Folin Phenol reagent. J. Biol. Chem., 193:265.

13) Mita, M. and Hall, P.(1982). Metabolism of round spermatids: Lactate as preferred substrate. Biol.Reprod. 26: 445-455.

14) Nakamura, M., Okinaga, S., Aria, K. (1984). Metabolism of round spermatids: evidence that lactate is preferred substrate. Am. J. Physiol. Reprod. 30: 1187 1197.

15) Ornstein, L. (1964). Disc electrophoresis, I Background and theory. Ann. N. Y. Acad. Sci.121:321.

16) Pesce, A.J.; Fondy, T.P.; Stolzenbach, F.G. Castillo, F.; Kaplan, N.O. (1967). The comparative Enzymology of lactic acid dehydrogenase III. Properties of the H4 and M4 enzymes from number of vertebrates. J.Biol. Chem. 242: 2151-2167.

17) Pillai, M.M., and Walvekar, M. V. (2002) :Effect of sialoadenectomy on thetestis and spermatogenesis. Ind. J. Comp. Anm. Physiol. 20: 8-13.

18) Pillai, M.M., and Walvekar, M. V. (2005): Effect of isoproterenol administration on testis and spermatogenesis. J Ecophysiol Occup Hlth, 4: 162-165.

Animals Age in days LDH concentration

't' value Statistical significance

Control

Sublingualectomised

Mother's male

Sialoadenectomised

Mother's male


Mother's male

90

90

90

90

5.24 0.4242

4.69 0.4678

3.97 0.6320

3.58 0.4320

±

±

±

±

2.3843

3.6509

7.4989

P< 0.01

P< 0.01

P< 0.001




+ S.D.

Copyright 2010 BioMedSciDirect Publications IJBMR - All rights reserved.

ISSN: 0976:6685.

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2564

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