Diyala Journal of Agricultural Sciences, 10(Special issue): 35-46, 2018 Hashim et al.

1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018

http://www.agriculmag.uodiyala.edu.iq/

PATHOLOGICAL STUDY AND GENETIC DIAGNOSIS OF

VINBLASTINE EFFECTS ON DNA OF OVARY AND TESTES IN

MOUSE

Muna Sachit Hashim1,4 Suad Abdul-Kareem Mohammed 1

Rabab Abd Almeer Naser2 Thikra Abdulla Mahmood3 Taghreed Jabbar Humadi1

1Dept. of Pathology, College of Vet. Med., University of Baghdad, Iraq.

2 Dept. of Anatom y, College of Vet. Med., University of Diyala, Iraq.
3 Dept. of Communit y Medicine , College of Medicine , Kufa Universit y, Iraq.

4Corresponding author: sachitmuna@yahoo.com

ABSTRACT

The present study was designed in order to identify the effects of

vinblastine on ovary and testes and then on fertilization capacity and pregnancy

outcome in white mice, fifty mixed mice were used, 10 treated male were mated

with 10 untreated female, 10 untreated male were mated with 10 treated female,

and (10) mixed mice as control. Results of male test showed decline in sperm

count with spermatocytes' morphological deformity. The results of molecular

technique applied on testicular section and ovarian section, showed that green

signals, indicating the damage on TK (11qE2)/XY gene on chromosome of

sperm and ova, red signal indicate intact gene yellowish signals indicated

translocation on the same gene (mutation). Pathological findings showed marked

loss of spermatogenesis (Azoospermia) with damage in testes' tissues; fibrosis of

ovarian stroma with degenerative changes on ovary and evidence of early

embryonic death with absence or low fertility.

Key words: Vinblastine, mouse, DNA, ovary, testes.

INTRODUCTION

Vinblastine belongs to a group of medications known as vinca alkaloids and

is obtained from the plant vinca rosea. Vinca alkaloids act as antimicrotubule

agents that block mitosis by arresting cells in the metaphase. In Veterinary

medicine, this agent represents the treatment of choice for canine transmissible

venereal tumor (CTVT). The treatment is effective for 90% of CTVT cases, by

intravenous (IV) administration, at the dose of 0.5-0.7 mg m-2 of body surface,

once a week (Withrow, 2001). Vinblastine is safe for most patients, but potential

side effects can occur, as gastro- intestinal alterations, myelosuppression and

extravasation injury (Said, 2009) vinca alkaloids caused neurotoxicity involve

peripheral, autonomic and central neuropathy (Graf, 1996). Neurotoxicity can

persist for months after discontinuation of therapy in some patients, and in rare

cases may be disabling (Egbelakin, 2011). Infants and embryos are at a higher

Diyala Journal of Agricultural Sciences, 10(Special issue): 35-46, 2018 Hashim et al.

1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018

http://www.agriculmag.uodiyala.edu.iq/

risk for experiencing Vinblastine-related neurotoxicity (Haskell, 1995).

Peripheral neuropathy is the most common type of neuropathy and develops in

almost all patients (McEvoy, 2004). Loss of deep tendon reflexes, peripheral

paresthesia, pain and tingling can occur. If therapy is prolonged or high doses

are administered, wrist and foot drop, ataxia, a slapping gait and difficulty in

walking can occur. Cranial nerve toxicities may lead to vocal cord paresis or

paralysis (hoarseness, weak voice), ocular motor nerve dysfunction (ptosis,

strabismus), bilateral facial nerve palsies, or jaw pain. Severe jaw pain can occur

within a few hours of the first dose of Vinblastine. Autonomic neuropathy

results in constipation (which can be severe), abdominal pain, urinary retention

and paralytic ileus. Central neuropathy includes headache, malaise, dizziness,

seizures, mental depression and psychosis (Perry, 2001).

Vinca alkaloids effects fertility

Chemotherapeutic agents can initiate accelerates the reduction of the

primordial follicle pool, induces ovarian atrophy and further a destruction of

vascularization of the ovary which leads to ovarian damage and risk of

premature ovarian failure, Follicular apoptosis and cortical fibrosis of ovaries

are also assumed effects related to treatment with chemotherapy (Meirow,

2010). Vinblastine therapy in female mouse caused defects in primary and

secondary follicles and corpus lutetium with in depression follicular stimulating

hormone (FSH) and luteinizing hormone (LH) and estrogen and progesterone

(Al-Ahmed, 2010). Vinca alkaloids crossed the blood-Placenta barrier to

produce cellular changes might play an important role in initiating growth

defects and dead of embryo (Joneja, 1969 ; Hejazi, 2012). Vinca alkaloids

reduced fertilization ability of male mice as results of DNA damage of sperm

and so lead to sterile mating or pseudo-dominant-lethal effects. Researcher

found that mice with vincristine treated father had a mutation rates up to twice

that of mice with untreated fathers notably, these mutations were present in

DNA inherited from both treated father and untreated mother, what this likely

means according to the researches measures is that chemotherapy induces

epigenetic changes in the sperm. Epigenetic Changes do not affect underlying

DNA sequence but they alter chemical tags that control how genes are expressed

this in turned to genome destabilization in the offspring, following sequence

mutation to arise in DNA from the mother or father. The exact mechanism for

genome destabilization and how it's inherited is unknown (Sarah, 2012).

Suggesting impairment of epididymis function particularly concerning sperm

maturation and endocytosis removal of contents of the cytoplasm droplets and

Diyala Journal of Agricultural Sciences, 10(Special issue): 35-46, 2018 Hashim et al.

1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018

http://www.agriculmag.uodiyala.edu.iq/

dead sperm (Averal, 1996). Male mouse treated with Vinca alkaloids for four

separated period 2, 4, 6 and 8 weeks suffered from gradually loss of testes

weight, sperm count, spermatocytes abnormalities with sever histopathological

lesions on testes tissue represented by loss of spermatogenesis and dead sperm

from apoptosis with inflammatory reaction further to necrosis (Al-Aamery,

2013).

MATERIALS AND METHODS

Fifty mixed mice were kept in animal house of Veterinary Medicine

College in Baghdad and fed on pellet for laboratory animal and provide with tap

water in special tubes, 10 male and 10 female were injected with Vinblastine I/P

(0.1 mg 10 gm-1 b.w) (Al-Aamery, 2013).

For one month (4 weekly injection) and mated with 20 mixed untreated

mice in separated groups. Other 10 mixed mice remained as control. After

scarified animals, sperm specimens collection directly from epididymis and

direct seminal fluid analysis was done (Al-Aamery, 2013).

(Cooper, 2010). Testes and ovary section were prepared on a charge slides

for molecular examination by using of FISH procedure to identify DNA damage

on spermatocytes according to (Al-Aamery, 2013 ; Wyrobek, 2005 ; Galina,

2010). Tissue specimen's testes and ovary were prepared for paraffin embedded

sectioning and histopathological processing with staining methods was done

(Slaoui, 2011).

RESULTS AND DISSCUSION

Results of fertility parameters in male and female

The results of seminal fluid analysis showed in table 1, that there was rapid

decrease in spermatocytes count with evidences of morphological deformities

and loss of testes weight. The results of molecular test showed in table 2 that

there was gradual and marked damage in DNA of sperm after 1st week of

treatment with Vinblastine and be more evident after 4th week of treatment. The

results of fertility parameters in females' mouse after treatment with Vinblastine

showed in table 3, that there was loss of ovarian weigh with bad pregnancy

outcome and visible loos in newborn with premature death of fetuses.

Diyala Journal of Agricultural Sciences, 10(Special issue): 35-46, 2018 Hashim et al.

1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018

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Table 1. Male fertility test

Group and

treatment time

n=10

Sperm count

± s.d 6×10

Abnormal

spermatozoa % ± s.d

Testes mean

weight (mg)± s.d

1- 1st week 5.18±1.21A 22.5±10.1C 361±25.6 a

2- 2nd week 4.30±0.63B 30.1±11.4C 302±35.6 B

3- 3rd week 2.16±1.43B 47.5±11.7C 202±38.0 B

4- 4th week 1.28±0.63B 61.2±17.3C 108±33.6 B

5- Control 6.15±1.22 13.4±1.04 422±25.6

Table 2. Molecular test for detection of DNA damage in ovary and testes

Group and

treatment

time n=10

Percentage of spermatocytes'

DNA damage ( (% ±s.d) with 3

score (Red, Green, Yellow) in

ovary

Percentage of DNA ovary (%

±s.d) with 3 score (Red, Green,

Yellow) in testes' tissue

st week1-1
Score1 score 2 score 3

6 2 2 a

score1 score2 score

3 4 5 a

nd week2-2 3 3 4 a 2 4 5 a

rd week3-3 2 3 5 a 1 6 3 A

th week4-4 0 2 8 A 0 1 9 B

5- Control 8 2 0 9 1 0

Differences from control are assessed by analysis of variance. Significant at: a: p˂0.05; A: p˂0.01;

B: p˂0.001 and assessed by the x2 test C: p˂0.001. Damage on DNA assed as: Score 1 (0-20%); score

2(20-50%); score 3(50- above 95 %).

Table 3. Females tested: for pregnancy outcome

Fertility

Dead

fetus

Live

fetuses

Pregnant

female

ovary mean

weight

(mg)±s.d

Group and

treatments'

time n=10

62% a 22% 78% 8 (80%) 430 ± 12.5 1-1st week

33% a 44% 56% 6 (60%) 333 ± 0.9 2- 2nd week

12% a 59% 41% 3 (30%) 187 ± 2. 6 3- 3rd week

2.2% a 88% 22% 1 (10%) 102 ± 1.25 4-4th week

95% a 5% 95% 10(100%) 545 ± 11.6 5-cotrole

Differences from control are assessed by analysis of variance. Significant at: a: p ˂ 0.05.

Vinblastine caused early loss of spermatogenesis with incidence of sperm

defects and heavy drop in testes weight after few week of treatment that’s'

proved and agreed with Al-Aamery (2013) and Dobrzynska (2003) in the same

family of drug uses. As well as agreed with Marvin (2013) who said that’s

chemotherapy causes reduction of sperm counts often to azoospermic levels that

may persist for several years or be permanent. Females of mouse suffered from

ovarian loss of weight due to ovarian hypoplasia with increase percent of

immature deliver baby with dead or defected fetuses that’s' lead to pad

Diyala Journal of Agricultural Sciences, 10(Special issue): 35-46, 2018 Hashim et al.

1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018

http://www.agriculmag.uodiyala.edu.iq/

pregnancy outcome and this agreed with (Hejazi, 2012). Who said that the

similar family drug vincristine has teratogenicity effects on the fetus as well as

clinical effects. Also agreed with studies based on the penetration of the drug

through blood-fetus and the cause of birth defects (Aigraina, 2005 ; Sadler,

2000).

Results of microscopic appearance

1-Molecular examination: was done by the application of florescence's in situ

hybridization (FISH) method and procedure on mouse samples testes and ovary

according to (Al-Aamery, 2013). Green signals in testes and ovary represented

the damage in site of TK (11qE2) / XY gene of sperm; Yellow signals refer to

mutation or translocation in the gene. Red signal represented the controls'

sample Figure 1 "A, B, C and D".

Fig. 1. A. testes section B. ovary section C. red signal of control D. yellow signal

The results of molecular test (FISH) examined the defect on DNA of

spermatocytes and ovary by calculating signals' percent referring to

chromosomal damage on TK (11qE2) /Y gene of sperm and ovary. Results

agreed with Al-Aamery (2013) who study the same effects of vincristine on

DNA'S sperm of mouse and with Wyrobec (2005) who use the same test.

2- Histopathological examination

Testes sections shows empty of seminiferous tubules (A) due to loose of

spermatogenesis with degenerative changes represented by hydropic

degeneration (B) with necrosis involve dead sperm and endothelial cells falls

into lumen (C) figure 2. In comparison with normal seminiferous filled tubules

(Figure 3).

A B

C
D

Diyala Journal of Agricultural Sciences, 10(Special issue): 35-46, 2018 Hashim et al.

1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018

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Fig. 2. Testes treated with Vinblastine showed empty seminiferous tubules (A) refer to

loss of spermatogenesis, with dilated and congested blood vessel (B) and necrosis (C)

(H&E 40X)

Fig. 3. Testes (control) showed spermatocytes filled seminiferous tubules (H&E 10X)

The pathological finding of females sections showed, that there's an

evidence of uterine fibrotic lesions involves endometrium (Figure 4. "A and B").

With atrophic and necrotic endometrial glands with glandular' epithelial lining

metaplasia and hyperplasia (Figure 5. "A and B"). Ovarian section of mouse

showed failure of ovulation at the end of 4 week of treatment with Vinblastine

with fibrotic lesions involves primary and secondary follicles within ovarian

stroma and hydropic degeneration (Figure 6).

Diyala Journal of Agricultural Sciences, 10(Special issue): 35-46, 2018 Hashim et al.

1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018

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Fig. 4. A. Uterus section of mouse showed cystic edematous degenerated endometrial

gland (A) with coagulative necrosis (B) (H&E 10X)

Fig. 4. B. Uterus section of mouse showed fibrosis (Black arrow) and comprised atrophic

degenerated glands (Red arrow) within endometrium (H&E 40X)

Diyala Journal of Agricultural Sciences, 10(Special issue): 35-46, 2018 Hashim et al.

1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018

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Fig 5. A. Uterus showed atrophic and necrotic endometrial glands (A) with glandular'

lumen epithelial lining (B) metaplasia and hyperplasia with papillary projection (H&E

10X)

Fig. 5. B. Uterus showed vacuolated glands (black arrow) with glandular lumen

distention with papillary projection from metaplasia and hyperplasia (Red arrow)

(H&E 10X)

Diyala Journal of Agricultural Sciences, 10(Special issue): 35-46, 2018 Hashim et al.

1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018

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Fig. 6. Ovarian showed early degenerative changes on follicles with failure of ovulation

(Red arrow) at the end of 4 week of treatment with vinblastine; fibrotic lesions (black

arrow) involves ovarian stroma with hydropic degeneration (A) (H&E 40X)

Vinblastine has toxic effects on mouse reproductive organs represents by

losing of spermatogenesis, vacuolation and necrosis within seminiferous tubules

and fibrosis in an advance stages, this effects increased with increased time of

treatments, and attributed to the pharmakinetic factor of drugs that’s caused

early pass of drug through blood testes barrier (BTB) and exerts it's toxicity as

soon as contact with testicular cells mainly leydig cells, sertoli cells, germs cells

so there's' evidence of oligospermia, azoospermia, and germinal aplasia. This

study's' results agreed with what found by (Al-Aamery, 2013 ; CC Lu,1979 ;

Roeser, 1998 ; Fairly, 1999). In female we found that s' vinblastine caused

severe fibrotic lesions within atrophic uterus and ovary, that's caused failure in

successful fertilized ova due to lose of mature follicles, lead to bad outcome of

pregnancy which increased with increased time of treatment. Beside the

immature parturition or early delivery with dead or defects fetuses. And this

agreed with Hejazi, 2012 ; Dobrzyńska, 2005 ; Karen, 2009).

CONCLUSION

A chemicals drug affects both histological and genetics structures of ovary

and testes.

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http://www.agriculmag.uodiyala.edu.iq/

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