Attaining Male Reversible Infertility through Modulation of Spermatogenesis by 2-(2″-chloro acetamido benzyl) - 3-(3′- indolyl) Quinoline or Indolyl Quinoline in Rats Rattus rattus

Research Article

Austin Andrology. 2016; 1(2): 1006.

# Attaining Male Reversible Infertility through Modulation of Spermatogenesis by 2-(2″-chloro acetamido benzyl) - 3-(3′- indolyl) Quinoline or Indolyl Quinoline in Rats Rattus rattus

Bhowal SK¹, Mahapatra P² and Sahu CR³*

¹Department of Zoology, Maulana Azad College, Kolkata, India

²Department of Zoology, Utkal University, India

³Department of Zoology, University of Kalyani, India

*Corresponding author: Sahu CR, Department of Zoology, University of Kalyani, Kalyani-741235, India

Received: June 22, 2016; Accepted: August 10, 2016; Published: August 12, 2016

## Abstract

The aim of the study was to investigate the fertility-regulating potential of the compound 2-(2″-chloroacetamidobenzyl) - 3-(3′-indolyl) quinoline or commonly called indolyl quinoline in male rats. Male rats of proven fertility were treated with the compound by oral gavages for 3, 6 and 12 weeks. Functional fertility, testicular, epididymal and seminal vesicular weight, epididymal sperm count as well as quantification of spermatogenesis process were made. Epididymis and testis samples were processed using routine techniques and prepared for light microscopy analysis in both treated and control series. Functional fertility was reduced significantly as revealed by a fall in fertility and pregnancy rate. The weight of the reproductive organs was reduced significantly. A reduction of sperm count and number of different types of testicular cells was observed. Histologically, the tested compound caused some disruption in both epididymis and testicular architecture, scanty spermatozoa and in number of differentiating spermatogenic cells. Histological alterations were assessed and the results were discussed according to the severity of the histological response made by the compound. Fertility and other effects were regained gradually after withdrawal of treatment. The results revealed from the study indicate reversible antifertility of the said chemical agent.

Keywords: Male Rattus rattus; Indolyl Quinoline; Spermatogenesis; Reproductive Organs; Anti-Spermatogenic; Reversible Antifertility

## Abbreviations

GnRH: Gonadotropic-Releasing Hormone; SRL: Sisco Research Laboratory; STM: Saline-Triton-Merthiolate; ANOVA: Analysis of Variance; NS: Not Significant; GPC: glycerol Phosphorylcholine

## Introduction

The population explosion is a global problem that poses significant threat to the quality of life in Third World countries. To combat this grave situation, effective family planning is required with equal participation of both males and females. An important section of reproduction is the fertility regulation by means of contraception and management of infertility [1]. Though some highly effective, acceptable and reversible contraception modalities have been developed as a contraceptive agent for the females, yet no or very little effective and reversible systemic method has been developed to date for males. Several prospective moves toward initiation of infertility using hormonal, chemical and immunological approach have been explored for a long time [2-5]. Induction of acute oligospermia or azoospermia is possible by using hormonal steroid or gonadotropic-releasing hormone (GnRH) agonist/ antagonist [6,7]. These methods are either irreversible or affect libido and secondary sex characteristics, and supplementary treatment is required appearing these methods cost-ineffective. Other way would be to find effective anti- spermatogenic agents that could rely on alternative means to the hypothalamic-pituitary gonadal axis, more particularly, compounds primarily designed for the purpose of other clinical use unrelated to contraception but unexpectedly revealed to have antifertility effect upon routine evaluation [8,9]. With the use of present-day knowledge of synthetic chemistry to synthesize different analogues and after careful pharmaco-toxicological surveillance, it is possible to achieve some useful products having no overt toxicity. Reversible antispermatogenic and antisteroidogenic properties of some traditionally used contraceptive have also been reported [10,11]. Nitrogenous heterocyclic compounds having antiflagellated or antiproliferative activity have been found to be antispermatogenic or vice versa [12]. Related compounds have been reported as antispermatogenic and have been projected as male contraceptive [2,3,13-15]. The imidazole group of compounds in clinical use against flagellated protozoa and anaerobic bacteria for genital tract infections of both men and women has been shown to have potential antifertility activity in male [16,17]. The compound 2-(2′-chloroacetamidobenzyl) - 3-(3′-indolyl) quinoline or indolyl quinoline contains indole, and quinoline moieties that match with all of the mentioned compounds [18,19]. The above information was the main impetus behind this effort of evaluating the fertility-regulating potential of the compound in male rats, including histomorphometric examination for better understanding of the spermatogenic process and testis function.

## Chemicals

All chemicals and reagents were of analytical grade and were procured from Merck India Ltd. Staining reagents were from SRL India Ltd., Mumbai, India. The tested chemical compound has been synthesised through the application of Friedel-Crafts reaction [20] the purity of the compound was verified [20] and the bioactivity was determined [21].

## Animals

All experiments on laboratory animals were performed as per the guidelines of the animal ethics committee of the University. Rats (Rattus rattus) from random bred colonies were housed on a fourteen hours light and ten hours dark cycle under standard husbandry conditions (temp. 220 C ±20 C, relative humidity 55 % ±5%) and were provided standard pellet food and sterile water ad libitum.

## Design of experiment, mating and fertility tests

Six different groups consisting of 5 animals in each group were allocated from a population of 60 male rats, where 50% of the total was considered as control without any treatment. The test material was administered orally by gavages to different groups of rats for 3, 6, and for 12 weeks at a dose of 250 mg/kg of body weight (5g/kg per day did not cause lethality, unpublished observation). The animals of one particular groups of a specific treatment were maintained for a period of another 3, 6 and 12 week each after discontinuation of the specific compound to find out any recovery of the effect of the test compound. Mating schedules for the experiment were planned in such a way, so that the infertility effect by the compound or the reversibility effect after discontinuation of the compound can be scrutinized accurately while monitoring their health and weight throughout the experimental period. Further the males were kept in a cage with the cyclic females (at pro estrous stage) as 1:1 ratio to observe any copulation plug or the presence of spermatozoa in the vaginal smear on the next morning and in successful mating it was considered as Day -1 of pregnancy. Accordingly, the percentage of successful mating was considered with the number of sperm positive females per number of exposed female. The so called successful mating females were separated on next morning while food and water was given ad libitum.

On day 10 of gestation, laparotomy of female rats was performed under anesthesia and their ovaries were excised, uteri were exposed including counting of corpora lutea, implantation sites and number of normal live fetuses was done.

Fertility rates were calculated by the percentage of implantation sites per number corpora lutea (representing the number of eggs ovulated) and pregnancy rate was considered to be the number of viable fetuses per female mated.

## Reproductive organ weight

The males (both treated and the controls) were sacrificed after the completion of treatment schedule. The testes, epididymides and the seminal vesicles were excised, cleaned, blotted free of blood, weighed and preserved or utilized for further histological and morphological analysis.

## Counting of sperm

The caudal region of epididymis was homogenized gently in a glass homogenizer in STM solution, containing 0.15 M sodium chloride, 0.05 % Triton X-100 v/v and 0.25M thimerosal. After thorough vortexing of the homogenate, one drop of the suspension was taken on the Makler counting chamber, covered with cover slip and observed under light microscope [22]. Sperms of four rows of ten squares of Makler chamber were counted. The process was repeated five times for each suspension and the average was taken and expressed per grams of epididymis.

## Epididymis

Epididymides of five rats per group were fixed in Bouin’s fixative, processed for histological sectioning, embedded in paraffin, and 6-μm sections were cut in a microtome and stretched on clean, grease-free glass slides. Deparaffinised sections were then processed for staining with eosin and hematoxylin and observed under microscope.

## Testis

The tunica albugenia of one of the two testes was nicked at both end after immersing in Bouin’s fixative for 24 hours and continued the fixation process for another 24 hours. Tissues were dehydrated in ethanol following several changes in upgrades of alcohol, thereafter, embedded in paraffin for section cuttings and microscopic studies. The tissue slides were processed for staining through eosin - hematoxylin and finally photomicrographs were taken.

## Quantification of spermatogenesis

Spermatological cells were quantified according to the method of Leblond and Clermont [23]. The relative number of each germ cell type at stage VII/VIII of the cycle of seminiferous epithelium, i.e., spermatogonia, Sertoli cells, spermatocytes and spermatid (Steps 7 and 19) was counted. The nuclear diameters of the different germ cells (Step 19 spermatids) were measured using Leitz micrometers. At least 20 round tubular cross sections were counted in each rat. All crude counts were corrected for difference in nuclear diameter by Abercrombie’s’ formula [24].

If P is the average number of nuclear points per section, A is the crude count of number of nuclei seen in the section; M is the thickness (in μ) of the section, and L the average length (in μ) of the nuclei, then

$\text{P=A}\frac{\text{M}}{\text{L+M}}$

## Statistical analysis

Results were expressed as mean ±standard deviation of mean of five repeated determination for 5 rats in each of the 6 groups of rats. The significance of difference in the mean data obtained were analysed and were compared using one-factor ANOVA. Values were considered significant when P<0.05 or less.

## Fertility status of the male rats

The fertility status of the female rats mated with a normal male rat not underwent any treatment showed a normal implantation site (Figure 1A). A significant reduction related to time of exposure in functional fertility as indicated by rate of pregnancy was noticed due to the introduction of the compound. The female rats mated with the compound treated male for 3, 6 and 12 weeks showed an average numbers (Figure 1B,1C,1D) of normal implantation sites as 4.8, 1.4 and 0.0 respectively (Table 1), whereas the females mated with control males showed number of implantation site 12.7 (Figure 1A). While the fertility rate for the control was 100% the treated group of rats showed a value ranges between 75% and 80% (Table 1). Thus, pregnancy rate of treated female animals showed decreased value compared to control group animals. Within 6 weeks of withdrawal of treatment a gradual recovery of fertility was noticed (Figure 1E,1F).