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INTRODUCTION
The unprecedented growth of the global population represents one of the most significant challenges of the modern era. Overpopulation has emerged as a critical global concern, as it directly or indirectly contributes to nearly all major worldwide problems. Issues such as poverty, malnutrition, depletion of natural resources, environmental pollution, global warming, loss of biodiversity, and even armed conflicts are exacerbated by population pressure. Consequently, the regulation of human fertility through effective limitation strategies has become one of the most urgent biosocial and medical challenges confronting humanity today (Hoesl et al., 2005).
Current statistics indicate that the global population has reached approximately 8.2 billion in 2024 and is projected to peak at around 10.3 billion by the mid-2080s. At present, India, with a population of about 1.38 billion, is the second most populous country in the world. Furthermore, projections suggest that India is expected to surpass China and become the world’s most populous nation by 2027 (Bhardwaj et al., 2025).
The current scenario necessitates the development of novel contraceptive options that are simple, safe, reversible, and cost-effective, and that are broadly acceptable across diverse cultures, religions, and racial groups worldwide. The availability of such methods would encourage greater adoption by couples, thereby contributing significantly to the reduction of population growth rates (Anderson and Baird, 2002; Page et al., 2008).
There are multiple target sites in the male reproductive physiology for functional disruption in order to develop novel male contraceptives. These include suppression of spermatogenesis in the testes, impairment of sperm maturation within the epididymis, prevention of sperm transport to the female reproductive tract, interference with sperm capacitation, and disruption of sperm functions essential for normal fertilization (Bhardwaj et al., 2025).
Extensive research has been conducted in the field of male contraception. Various hormonal approaches, including androgens, progestogens, estrogens, and GnRH agonists or antagonists, alone or in combination as well as immunological and chemical agents, have been investigated. However, none of these methods has yet proven to be completely effective, safe, and free from adverse side effects (Page et al., 2008; Tulsiani and Abou-Haila, 2008).
In recent decades, considerable attention has been directed toward medicinal plants documented in classical Materia Medica, Ayurvedic literature, and folk medicine for their potential antifertility effects in males. Numerous plant species have been systematically screened worldwide, revealing promising leads for the development of plant-based male contraceptives (Unny et al., 2003; Obuewu et al., 2011).
The development of novel fertility-regulating drugs derived from medicinal plants represents an attractive proposition, as plant-based contraceptives are generally more acceptable due to their cost-effectiveness, easy availability, and comparatively fewer adverse side effects. Traditional medicine continues to serve as the primary healthcare system for approximately 70-80% of the global population, particularly in developing countries. Medicinal plants constitute a rich source of diverse bioactive phytochemicals that may act either individually or synergistically to exert antifertility effects, offering valuable leads for drug development based on phytochemical investigations (Rates, 2001; Gelani and Rehman, 2005).
Pongamia pinnata (Linn.) Pierre is a medium-sized, glabrous tree commonly known as Karanja in Hindi and Indian beech in English. It is an important medicinal plant predominantly found in the tidal forests of India and has long been used in the traditional Indian system of medicine for the treatment of bronchitis, whooping cough, rheumatic arthritis, and diabetes. Numerous pharmacological investigations have demonstrated that P. pinnata exhibits a wide spectrum of biological activities, including antioxidant, antimicrobial, antiparasitic, anti-inflammatory, anticonvulsant, antidiabetic, antihyperammonemic, cytotoxic, anthelmintic, insecticidal, and immunomodulatory properties (Akram et al., 2021; Kyatham et al., 2024).
Phytochemical studies of P. pinnata have revealed the presence of a diverse array of bioactive constituents. Flavonoids and their derivatives represent the most commonly isolated compounds, including flavones, flavans, and chalcones. In addition, several other classes of phytochemicals such as sesquiterpenes, diterpenes, triterpenes, steroids, amino acids, disaccharides, fatty acids, and ester compounds have been identified (Muquarrabun et al., 2013; Kyatham et al., 2024). Notably, seven flavonoids; pongaflavone, karanjin, pongapin, pongachromene, 3,7-dimethoxy-3′,4′-methylenedioxyflavone, millettocalyxin C, and 3,3′,4′,7-tetramethoxyflavone have been isolated from the stem bark of P. pinnata (Yin et al., 2004; Muquarrabun et al., 2013).
Despite its prominent status in Ayurvedic literature and traditional medicine, there is a paucity of scientific data regarding the effects of P. pinnata on male reproductive function. Therefore, the present study was undertaken to evaluate the potential contraceptive efficacy of the 50% ethanolic stem bark extract of P. pinnata in male rats.
MATERIALS AND METHODS
Plant material and Method of extraction
The plant material was collected from the University of Rajasthan Campus and authenticated at the Herbarium, Department of Botany, University of Rajasthan, Jaipur. A voucher specimen of the plant has been deposited in the herbarium (RUBL-20255). The collected stem bark was shade-dried, ground into coarse powder, and extracted with 50% ethanol using a soxhlet apparatus for 36 hr at 60-80°C. The crude extract obtained was filtered and concentrated to dryness under reduced pressure at low temperature (40°C). The resulting viscous brown residue was then suspended in an appropriate volume of distilled water and used for experimental administration.
Experimental animals
Adult, healthy male albino rats (Wistar strain) of proven fertility were used in the present study. The rats were housed in groups in polypropylene cages (12" × 10" × 8") under standard laboratory conditions, with a 14 hr light: 10 hr dark cycle and a controlled temperature of 22 ± 3°C. Animals were provided with water and a standard laboratory diet (Lipton India Pvt. Ltd.,) ad libitum, and occasionally supplemented with soaked grams and leafy vegetables. Animal care and handling were conducted in accordance with the guidelines of the Indian National Science Academy (INSA, 1992, New Delhi, India) for the maintenance and use of laboratory animals. Experimental procedures were approved by the Institutional Animal Ethics Committee (Department of Zoology, University of Rajasthan, Jaipur).
Experiment design
Rats of nearly equal size; weight (170-210 g) were selected and than randomly divided into four groups each having seven rats and treated as follows:
Group I: Control rats treated with vehicle (distilled water, 0.3 mL/rat/day) for 60 days.
Group II: Rats treated with P. pinnata extract (100mg/kg b.wt./day) orally, suspended in distilled water for 60 days.
Group III: Rats treated with P. pinnata extract (200 mg/kg b.wt. /day) orally, suspended in distilled water for 60 days.
Group IV: Rats treated with P. pinnata extract (300 mg/kg b.wt. /day) orally, suspended in distilled water for 60 days.
24 hr after administration of the lost dose, all the overnight fasted rats from various treated groups were sacrificed under ether anesthesia. Blood samples were collected by cardiac puncture. The blood sample was allowed to clot at 37°C and the serum was separated by centrifugation and stored at-20°C for biochemical analysis.
Testicular weight
Testicular weights of all rats were recorded at the end of the treatment. Testes and other reproductive organs were dissected out, cleaned of adherent fat and blood clots, and weighed individually on a digital electronic balance. One half of the reproductive tissues was fixed in bouin’s fluid for histopathological examination, while the remaining half was stored at -20°C for biochemical estimations in future.
Serum testosterone
Serum level of testosterone hormone was measured by Chemiluminiscence-CIA by autoanalyzer.
Histological study
For histological observation, bouin's fixed tissues (testis) were washed in water to remove excess of fixative, dehydrated in graded series of alcohol, cleared in xylene, embedded in paraffin wax and sectioned at 5 µm and counter stained in eosin for the cytoplasmic contrast. Sections were observed for histopathological effects using a Nikon digital camera mounted on a light microscope.
Germ cell population and Histomorphometric analysis
Quantitative evaluation of germ cells
Quantitative evaluation of germ cells were made using 50 round tubules per group selected randomly at X 400 according to method described by Leblond and Clermont (1952). Quantitative evaluation of spermatogonia, preleptotene, mid-pachytene spermatocytes: round and elongated spermatids were made in stage VII of seminiferous tubule cycle per cross section under X100 magnification. Counting was performed in 10 tubule cross section and mean was calculated.
The diameters of nuclei of various germ cell types were measured by mean of an ocular micrometer. A correction factor was used to obtain the actual numerical density of germ cells (Abercrombie, 1946).
Seminiferous tubule diameter
Measurements were taken from at least 40 tubular profiles per animal using a light microscope equipped with ocular micrometer caliberated with stage micrometer. Only round tubules were selected randomly in various areas of the section. Two diameters perpendicular to each other were measured at X 100 magnification and averaged and expressed as seminiferous tubule diameter.
Leydig cell area and nuclear diameter
The diameter of one hundred Leydig cell and their nuclei were measured on five sections from each testis with ocular micrometer at X 1000. The values were averaged and expressed as mean nuclear diameter of the Leydig cells.
Statistical analysis
All the data were calculated and statistically analyzed with SPSS\V.13.0 computer software package for Windows (SPSS Inc., Chicago, IL., USA). The data were expressed as Mean ± SEM and tested for variance. When variance was homogenous, one way Analysis of Variance (ANOVA) was applied out to determine the significance of observed differences, the test. An alpha probability of less than 5 % (p<0.05) was considered as statically significant. All other data were statistically analyzed with one way ANOVA for the comparison between the groups followed by Tukey’s multiple comparison procedure as a post hoc test.
RESULTS AND OBSERVATIONS
Testicular weight
Treatment with P. pinnata extract resulted in a significant (p < 0.05), dose-dependent reduction in relative testicular weight in all treated groups (100, 200, and 300 mg/kg body weight/day). The decrease was significant not only when compared with the control group but also among the treated groups themselves (Figure 1).
Serum testosterone
Serum testosterone levels in rats treated with P. pinnata extract exhibited a significant, dose-dependent decline compared with the control group, as well as upon multiple comparisons among the three treated groups (100, 200, and 300 mg/kg body weight/day). The mean serum testosterone concentration in control rats was 3.81 ng/mL, which decreased to 2.25 ± 0.11 ng/mL in the highest-dose treatment group (Figure 2).
Histopathology
The histological picture of the testis of control rats displayed normal process of spermatogenesis. The seminiferous epithelium showed characteristic tiered arrangement of all successive germ cell types. The tubular lumen was fully occupied by large number of healthy spermatozoa. The presence of large number of normal Leydig cells was also conspicuous in the interstitium (Figures 3A & 3B, Table 1).
| Treatment | Control | Pongamia pinnata | ||
|---|---|---|---|---|
| Parameter | 100 mg/kg b.wt. | 200 mg/kg b.wt. | 300 mg/kg b.wt. | |
| Spermatogonia | 6.92±0.42a* | 6.79±0.24 a | 5.94±0.66 a | 5.37±0.92 b |
| Preleptolene spermatocyte | 20.14±1.02 a | 19.42±0.97 a | 17.85±0.67 b | 16.57±0.75 c |
| Mid-pachytene spermatocyte | 23.92±0.94 a | 20.28±0.93 b | 19.27±0.65 c | 17.07±0.82 d |
| Round spermatid | 42.0±3.02 a | 33.28±1.88 b | 23.85±2.48 c | 15.71±1.04 d |
| Elongated spermatid | 34.66±1.88 a | 22.64±1.66 b | 16.0±1.04 c | 8.42±0.77 d |
| Seminiferous tubular diameter (µm) | 277.85± 9.93 a* | 244.85± 10.24 b | 237.00±9.00 c | 219.71±9.30 d |
| Leydig cell nuclear diameter (µm) | 6.94± 0.09 a | 6.22±0.11 b | 6.11±0.15 c | 5.82±0.15 d |
The histoarchitecture of the testis in rats receiving P. pinnata extract (100 mg/kg/day) showed degenerative changes. The diameter of seminiferous tubules was reduced (p<0.05). There was significant (p<0.05) decline in the number of round and elongated spermatids. The characteristic tiered arrangement was also disturbed. The number for spermatogonia and preleptotene spermatocyte remained unaffected. The Leydig cells showed atrophic changes (Figures 3C & 3D, Table 1).
The histoarchitecture of the testis in rats treated with 200 mg/kg/day dose of P. pinnata extract showed marked degenerative changes and disruption of spermatogenesis. The seminiferous tubules were reduced in size and showed disruption of normal epithelial organization. Quantitative determination of germ cells in seminiferous epithelium showed a decline in the numbers of preleptotene and pachytene spermatocyte (p<0.05), round and elongated spermatids (p<0.05). However, the number of spermatogonia remained significantly unaltered. Additionally, several cells with pycnotic nuclei could be identified. The lumen showed the presence of exfoliated germ cells, sperm debris and few spermatozoa. The Leydig cells were shrunken and showed atrophic changes (Figures 3E & 3F, Table 1).
Atrophic changes in the testis were more pronounced at 300 mg/kg/day dose of the extract. The diameter of seminiferous tubules was further decreased. There was significant decrease in the numbers of spermatogonia (p<0.05) preleptotene and pachytene primary spermatocytes, (p<0.05), round (p<0.05) and elongated spermatids (p<0.05). The lumen contained tail reminants of degenerating spermatozoa. Leydig cells also showed marked degeneration, decrease in nuclear diameter and decline in numbers (Figures 3G & 3H, Table 1).
It appears that P. pinnata extract induced dose dependent degenerative, atrophic changes and disruption of spermatogenesis in treated male rats.
Germ cell population and Histomorphometric analysis
The testicular germ cells population i.e. spermatogonia, preleptotene spermatocyte, pachytene spermatocytes, round, elongated spermatids counts/seminiferous tubule cross section, Seminiferous tubule diameter and Leydig cell nuclear diameter are presented in Table 1.
The control value of spermatogonia in per seminiferous tubule was 6.92 ± 0.42. After 60 days of the P. pinnata extract treatment a non-significant decline in mean spermatogonia numbers was observed in both 100 mg/kg and 200 mg/kg/ b.wt/day dose groups. However, in 300 mg/kg b.wt. dose group of P. pinnata the decline was significant (p<0.05). The mean counts of preleptotene spermatocytes per seminiferous tubule cross section were 20.14±1.02. Treatment of P. pinnata extracts at three different doses (100, 200 and 300 mg/kg/ b.wt/day) showed dose dependent significant (p<0.05) diminution of preleptotene primary spermatocyte population when compared to control or the three groups compared with each other except in 100mg/kg groups of P. pinnata, where the decline was not significant. The population of pachytene spermatocyte in the seminiferous tubules was declined dose dependently (p<0.05) in rats treated with P. pinnata extract at three different doses. There was significant (p<0.05) dose dependent decline in the populations of round and elongated spermatids per seminiferous tubules cross section in all the groups of rats treated with P. pinnata extract for 60 days.
There was significant (p<0.05) dose dependent decrease in the mean diameter of seminiferous tubules in the rats treated with three different doses (100,200 and 300 mg/kg/ b.wt/day) of P. pinnata extracts as compared to control and also on multiple comparison test.
Treatment of P. pinnata extract at 100,200 and 300 mg/kg/ b.wt/day,dose levels for 60 days resulted in a significant (p<0.05) reduction of Leydig cell nuclear diameter when compared to control and also between three groups themselves.
DISCUSSION
Traditional medicine has long played a vital role in healthcare systems worldwide, particularly in India, where plant-based remedies form the foundation of indigenous systems such as Ayurveda. The widespread use of medicinal plants is largely attributed to their perceived low toxicity and the long history of human exposure. In this context, the present investigation was undertaken to evaluate the antispermatogenic and antifertility potential of the medicinally important plant Pongamia pinnata in male Wistar albino rats.
In the present study, a dose-dependent decline in the relative weight of the testes was observed following administration of P. pinnata stem bark extract. Similar reductions in testicular weight have been reported earlier in animals treated with various plant extracts possessing antispermatogenic and antiandrogenic activities (Purohit and Bhagat, 2004; Jain and Meerwal, 2016). The decrease in testicular weight may be attributed to the loss of spermatids and spermatozoa, reduction in seminiferous tubular diameter, and inhibition of steroid biosynthesis by Leydig cells (Nandi et al., 1999; Jana et al., 2006; Jain and Meerwal, 2016).
A significant dose-dependent decrease in serum testosterone levels was also recorded in rats treated with P. pinnata extract. This reduction in androgen levels may result from a direct inhibitory action of the extract on Leydig cells or indirectly through interference with the hypothalamic-pituitary-gonadal axis, leading to reduced gonadotropin secretion and impaired androgen biosynthesis. The observed decline in testosterone levels may be due to the adverse effects of phytoconstituents present in the extract on steroidogenic activity. Phytochemical studies of P. pinnata have demonstrated the presence of flavonoids, flavones, and terpenes, compounds known to exhibit antiandrogenic as well as estrogenic properties (Bandivdekar and Moodbidri, 2002; Jain and Meerwal, 2016). It is well established that estrogens inhibit androgen biosynthesis in Leydig cells, thereby affecting normal spermatogenesis (Abney, 1999; Dias et al., 2014).
Histopathological examination of the testis is considered the most sensitive endpoint in the evaluation of male reproductive toxicity (Creasy, 2001). In the present study, histological analysis revealed marked dose-dependent degenerative and atrophic changes in the testes of rats treated with P. pinnata extract. The seminiferous epithelium showed exfoliation and disorganization of germ cells, along with a significant reduction in the number of preleptotene spermatocytes, pachytene spermatocytes, round and elongated spermatids, and spermatozoa within the lumen. In the higher dose groups, depletion of spermatogonia was also evident. Additionally, the seminiferous tubules exhibited reduced diameter, and Leydig cells showed signs of atrophy.
These histological alterations are likely a consequence of decreased serum testosterone levels, which are essential for maintaining normal spermatogenesis and the structural integrity of seminiferous tubules (Sharpe et al., 1988). Seminiferous epithelial sloughing may result from damage to Sertoli cells and disruption of intercellular bridges. Moreover, it is well documented that a decline in intratesticular testosterone induces germ cell apoptosis, further contributing to impaired spermatogenesis (Kim et al., 2001; O’Donnell et al., 2006; Yang et al., 2006). Furthermore, the present observations are consistent with several studies reporting testicular lesions, reduced germ cell populations at various stages of development, and Leydig cell atrophy following treatment with plant extracts exhibiting antiandrogenic activity (Jain and Ali, 2007; Nusier et al., 2007; Yakubu et al., 2007; Jain and Meerwal, 2016).
Phytochemical investigations have shown that P. pinnata stem bark extract is rich in phenylpropanoids, flavonoids, and pongamones (Kitagawa et al., 1992; Yin et al., 2004; Li et al., 2006). Flavonoids and isoflavonoids such as rutin and quercetin are well-known phytoestrogens and have been reported to exert antifertility and antiandrogenic effects in male rats (Bhargava, 1989; Das et al., 2004). Farnsworth et al. (1975) also reported the antifertility potential of rutin and quercetin. Several estrogenic compounds that bind to estrogen receptors in male reproductive organs have been shown to induce similar degenerative changes and suppression of spermatogenesis through a decline in serum testosterone levels (Boockfor and Blake, 1997; Jana et al., 2006; Yang et al., 2007).
Overall, the results of the present investigation strongly suggest that the phytoconstituents present in P. pinnata stem bark extract exert deleterious effects on male reproductive function, primarily through antiandrogenic and estrogenic mechanisms, leading to impaired spermatogenesis and reduced fertility.
CONCLUSION
Based on the findings of the present investigation, it can be concluded that the phytoconstituents present in the 50% ethanolic stem bark extract of Pongamia pinnata, acting either individually or synergistically, produced marked adverse effects on the testes of male Wistar rats. Administration of the extract led to pronounced degenerative changes in testicular histoarchitecture, along with significant impairment of spermatogenesis. Taken together, these alterations reflect a compromised functional integrity of the testes, which ultimately resulted in a decline in male fertility.