Dietary fluted pumpkin seeds induce reversible oligospermia and androgen insufficiency in adult rats

ABSTRACT

Some components of the human diets are believed to be promising male contraceptive agents. The present study examined the antispermatogenic efficacy, reversibility and toxicity of fluted pumpkin seed-supplemented diet (DFPS) in adult male Wistar rats. Adult rats were given DFPS at 2.5, 5 and 10% for 60 days followed by 60 days post-treatment period. The control animals received normal standard rat diet not supplemented with fluted pumpkin seeds. The sperm quality variables, testosterone and follicle-stimulating hormone (FSH), oxidative status of the testis, steroidogenic enzymes and gamma-glutamyl transferase (γ-GT) activities and the histology of the testis were determined to evaluate the anti-fertility activity of fluted pumpkin seeds. Treatment of animals with DFPS at 5% and 10% resulted in decreased serum and intra-testicular testosterone and FSH concentrations. This effect was associated with decreased activity of 17β-hydroxysteroid dehydrogenase (17β-HSD), increased testicular oxidative stress and poor sperm quality in the 10% diet group. After 60 days DFPS post-treatment, intra-testicular 17β-HSD and γ-GT activities, FSH and testosterone levels recovered to control values. Furthermore, poor sperm motility, count, morphology and viability as well as severe loss of spermatogonia and other matured epithelial germ cells and Sertoli cells observed especially in the 10% DFPS-treated animals reverted to nearly control values 60 days after withdrawal of treatment. Dietary fluted pumpkin seeds may selectively act on the epithelial germ cells, possibly mediated via Sertoli cells, leading to oligospermia, oxidative damage and androgen insufficiency. The reversibility of these effects to near normal levels after withdrawal of treatment justifies further consideration of DFPS as it may be an effective and readily reversible agent that meets the required criteria of a male contraceptive agent.

Abbreviations: GC-MS: gas-chromatography mass spectrophotometry; MPO: myeloperoxidase; NO: nitric oxide; DFPS: dietary fluted pumpkin seeds; DFPS (REV): DFPS post-treatment; MDA: malondialdehyde; SOD: superoxide dismutase; CAT: catalase; GSH: glutathione; 3β-HSD: 3β-hydroxysteroid dehydrogenase; 17β-HSD: 17β-hydroxysteroid dehydrogenase; NAD: nicotinamide adenine dinucleotide (oxidized); NADH: nicotinamide adenine dinucleotide (reduced); ITT: intra-testicular testosterone; FSH: follicle-stimulating hormone; FPS: fluted pumpkin seeds; NIST: National Institute Standard and Technology; Av: absolute volume; Ac: cross-sectional area; ST: seminiferous tubules; γ-GT: gamma glutamyl transferase.

KEYWORDS:

• Fluted pumpkin seeds
• sperm quality
• hormones
• diet
• oligospermia

Introduction

Potent and innocuous forms of contraception suitable for different couples and diverse cultures are crucial for family planning (Chauhan and Agarwal 2010; Plana 2017; Ain et al. 2018). Obviously, numerous fertility control efforts are aimed at women, and men have been asked to share in this responsibility (Amory 2016; Plana 2017). The call for men to be equal partners with women in fertility regulation has been slow due to limited acceptable contraceptive options (Plana 2017). More so, complications associated with existing male contraceptive options such as hormonal imbalance, epididymitis and semen leakage prompted the search for other methods of male contraception (Anawalt and Amory 2001; Kanakis and Goulis 2015; Ain et al. 2018). This led to considerable efforts in the formulations of hormone and non-hormonal dependent male contraceptives. Hormone dependent male contraceptives tend to influence the spermatogenic process via the suppression of hypothalamic-pituitary-testicular axis leading to infertility and reduced sperm count (Meriggiola and Pelusi 2006; Xie et al. 2017). Of these, testosterone enanthate and testosterone undecanoate suppresses the endogenous synthesis of testosterone and reduces spermatogenesis (Kanakis and Goulis 2015). This method was observed to promote undesired side effects such as lowering of high-density lipoprotein, hypertension, weight gain, and cancer (Anawalt and Amory 2001; Nieschlag et al. 2003; Kumar et al. 2012).

Although efforts to develop effective plant-derived male contraceptive agents have been undertaken, the progress in this area has been minimal. For instance, daily use of Tripterygium wilfordii extract elicited antifertility properties in animals and men by distorting sperm development and lowering sperm count but its use was associated with adverse side effects (Qian 1987; Herdiman et al. 2006; D’Cruz et al. 2010; Jing et al. 2017). Furthermore, gossypol obtained from cotton seed oil provoked antifertility effects in human and animal models through reduction in sperm quality and increase in sperm mortality, degeneration of the testis and disruption of spermatogenesis and steroidogenesis (Coutinho 2002; Santana et al. 2015; Lim et al. 2019). However, a reduction in serum potassium levels and permanent azoospermia was observed in volunteer patients taking gossypol (Coutinho 2002; Lim et al. 2019). Furthermore, the irreversibility of azoospermia, slow contraceptive effectiveness, and other undesired side effects associated with some plant-derived contraceptive agents have heightened the focus of most laboratories on the search of potential plant-based male contraceptives that satisfy the criteria of an effective male-contraceptive agent (Coutinho 2002; Jing et al. 2017; Verma et al. 2017; Ain et al. 2018; Lim et al. 2019).

Telfairia occidentalis Hook (Cucurbitaceae), commonly called fluted pumpkin, is an important dietary item and deep-rooted part of herbal medicine, especially in the eastern part of Nigeria (Akoroda 1990; Akwaowo et al. 2000). As food, the fleshy kernel obtained from de-shelled and boiled seeds of T. occidentalis is consumed as snacks while its fermented form is used as seasoning to flavor soup (Akoroda 1990; Saalu et al. 2010). It is now thought that some components of T. occidentalis seeds including alkaloids, tannins, oxalates, and erucic acid could provoke diverse biochemical, physiological and morphological outcomes (Saalu et al. 2010; Akang et al. 2011; Njoku et al. 2018).

With respect to gonadal functions, extract of T. occidentalis leaves can reduce testosterone, alter sperm quality and damage the testicular architecture of rats (Adisa et al. 2014). Furthermore, T. occidentalis leaf extract is reported to have significant amounts of tannins, alkaloids, and saponins reducing sperm count and motility and increasing the number of abnormal spermatozoa together with testicular oxidative stress (Saalu et al. 2010). On the contrary, the reports of Aghaei et al. (2014) suggested that diet supplemented with seed extracts of T. occidentalis known to contain fatty acids, iron and anthraquinones (Eseyin et al. 2018) improved sperm viability, motility, sperm count and normal sperm morphology as well as testicular architecture of rats exposed to a gonadotoxic agent. Spermatogenesis in rats lasts for 54 days (Foster et al. 2010; Abarikwu et al. 2017) and so treatment regimen of 4–6 weeks to study spermatogenesis as reported in Aghaei et al. (2014) might be less physiologically significant. Though the treatment duration in Saalu et al. (2010) was longer than the spermatogenic cycle for rats, the dose at which the testiculotoxic effects were observed were also too high to be encountered in most natural health foods. In our earlier study, we had supplemented rat standard diet with boiled seeds of T. occidentalis at doses of 10% (100 g/kg), 15% (150 g/kg) and 30% (300 g/kg) and evaluated their effect on the rat testis through the complete duration of spermatogenic cycle, and also observed degenerated seminiferous epithelium accompanied with loss of germ cells, as well as altered sperm quality (Njoku et al. 2018).

Therefore, the present work was designed to further evaluate the antifertility potential of dietary fluted pumpkin seeds (DFPS) in male rats at somewhat lower doses than was reported in our previous study, as well as its effect on the testis 60 days after withdrawal of treatment. The former would likely be encountered in most natural health foods

Results

Absolute and relative weight of testes and antioxidant defense and lipid peroxidation in the testes

In this study, changes in the absolute and relative weight of the testis of 2.5, 5 and 10% DFPS treated rats were not statistically different (Table 1). There was also no change in the final body weight of animals across all groups (data not shown).

Table 1. Testis weight, tubular diameter, epithelial height and tubular lumen of adult rats treated with fluted pumpkin seeds supplemented diet for 60 days and 60 days after withdrawal of treatment.

Groups	ATW (g)	RTW	TDi (µm)	SEH (µm)	TLu (%)
Control	2.38 ± 0.67	0.98 ± 0.28	125.03 ± 20.69	29.57 ± 6.74	18.78 ± 3.54
2.5% DFPS	2.01 ± 0.87	0.99 ± 0.45	111.84 ± 25.14	26.73 ± 5.91	21.15 ± 3.66
5% DFPS	2.18 ± 0.79	1.10 ± 0.43	134.14 ± 21.78	29.80 ± 4.50	29.80 ± 4.62
10% DFPS	2.25 ± 0.85	0.92 ± 0.36	87.08 ± 15.61*	7.36 ± 2.23*	77.23 ± 10.45*
2.5%DFPS (REV)	1.92 ± 0.47	0.82 ± 0.18	149.40 ± 17.84	30.07 ± 4.34	16.42 ± 4.38
5%DFPS (REV)	1.96 ± 0.61	0.89 ± 0.26	141.32 ± 23.81	23.01 ± 6.28	17.16 ± 6.82
10%DFPS (REV)	2.18 ± 0.79	0.89 ± 0.32	135.95 ± 23.69**	20.41 ± 4.22**	16.88 ± 5.04**

Each value represents the mean ± SD. *Significantly different from the control. **Significantly different from 10% DFPS (p < 0.05). (n = 5). Absolute Testis weight = ATW. Relative testis weight = RTW. Tubular diameter = TDi. Seminiferous epithelial height = SEH. Tubular lumen = TLu.

Consumption of fluted pumpkin seed-supplemented diet at all doses significantly (p < 0.05) increased the concentration of MDA compared with non-pumpkin diet group (Figure 1A). More so, DFPS at all administered doses elevated the activity of catalase in the testes which reached significant level (p < 0.05) at 10% compared with the untreated rats (Figure 1B). Exposure to 2.5 and 5% DFPS resulted in a significant decrease (p < 0.05) in the concentration of GSH while 10% DFPS exposure elevated the level of GSH in the testis compared with the control (Figure 1C). However, after the withdrawal of fluted pumpkin diet, the alterations in antioxidant status and lipid peroxidation were reversed in comparison to the initial treatment values.

Figure 1. Effects of dietary fluted pumpkin seeds (DFPS) on testicular oxidative stress markers of rats after 60 days treatment and 60 days post-treatment withdrawal. Oxidative stress pointers were quantified in the testes homogenates prepared in ice-cold phosphate buffered saline as described in the material and methods section. (A) malondialdehyde (MDA), (B) catalase, and (C) reduced glutathione (GSH). Data are presented as the mean ± SD (n = 5). *Versus control group, **versus 5% DFPS group, and ***versus 10% DFPS group (p < 0.05).

Effect of DFPS on the steroidogenic enzyme (3β-HSD and 17-βHSD) activities and γ-glutamyl transferase in the testis

The decrease of 3β-HSD activity in the testis of rats fed DFPS at all doses did not reach statistical significance versus the control values (p > 0.05). The activity of 17-βHSD was significantly decreased (p < 0.05) in the 10% DFPS group but remained unchanged in the 2.5 and 5% DFPS groups compared to control values. Following withdrawal of the DFPS treatment, the activities of 3β-HSD appeared to significantly increase in a dose-dependent manner but reached statistical significance only in the 10% DFPS (REV) animals. Similarly, 17-βHSD activity was significantly improved in the 10% post treatment group compared to the initial treatment values (p < 0.05; Figure 2A,B). It was also observed that animals treated with a fluted pumpkin seed-supplemented diet at 5% DFPS and 10% DFPS significantly (p < 0.05) increased the activity of testicular γ-GT in comparison with control values, although no significant change was observed in the 2.5% DFPS group (Figure 2(C)). Following 60 days of treatment withdrawal, the activity of γ-GT reduced in the 5% DFPS (REV) compared to the treatment group, though it remained unchanged in the 10% DFPS (REV).

Figure 2. Effects of dietary fluted pumpkin seeds (DFPS) on testicular steroidogenic enzyme and γ-glutamyl transferase activities of rats after 60 days treatment and 60 days post-treatment withdrawal. Steroidogenic enzymes and γ-glutamyl transferase were determined in testes homogenate prepared in ice-cold phosphate buffered saline as described in the material and methods section. (A) 3β-hydroxysteroid dehydrogenase (3β-HSD), (B) 17β-hydroxysteroid dehydrogenase (17β-HSD), and (C) γ-glutamyl transferase (GGT). Data are presented as the mean ± SD (n = 5). *Versus control group and **versus 10% DFPS group (p < 0.05).

Effect of DFPS on serum testosterone and follicle-stimulating hormone

There was significant decrease (p < 0.05) in serum and intratesticular testosterone observed in rats exposed to 10% DFPS (Figure 3(A,B)). Additionally, the decrease in serum testosterone in the 5% DFPS group did not reach a statistically significant level and also did not change significantly in the testis but there was marked increase in intratesticular and slight (insignificant) increase in serum testosterone in rats fed 2.5% fluted pumpkin seeds. The decrease in serum FSH in rats exposed to DFPS at 2.5%, 5% and 10% did not reach statistical significance compared to control values. In the testis, the increase in FSH in the 2.5% DFPS animals and slight decrease in the 5% and 10% DFPS groups compared to the control values did not also reach statistical significant values (p > 0.05). After treatment withdrawal, there was a significant decrease in intratesticular FSH in the 2.5% DFPS (REV) and an improvement in testis FSH in the 5% DFPS (REV) and 10% DFPS (REV) treated animals compared to corresponding initial treatment values. In the post-treatment groups, serum, FSH also decreased in the 2.5% DFPS (REV) animals but this decrease did not reach statistical significance compared to initial treatment group. However, serum FSH in the 5% DFPS (REV) animals have their levels higher but did not reach statistical significance level but remained unchanged in the 10% DFPS (REV) animals compared to the corresponding initial treatment (Figure 3C,D).

Figure 3. Effects of dietary fluted pumpkin seeds (DFPS) on testosterone (T) and follicle-stimulating hormones (FSH) of rats after 60 days treatment and 60 days post-treatment. Testosterone and follicle-stimulating hormone were quantified in the serum and testes homogenate with enzyme-linked immunosorbent assay (ELISA) kit. (A) serum T, (B) intra-testicular T (ITT), (C) intra-testicular FSH, and (D) serum FSH. Data are presented as the mean ± SD (n = 5). *Versus control group and **versus 10% DFPS group (p < 0.05).

Effect of DFPS on sperm quality, histopathology and testis morphometry

Treatment of animals with DFPS prompted a significant reduction (p > 0.05) in percentage motile sperms, caudal epididymal sperm numbers and sperm viability compared to the untreated group. However, the decrease in motile sperms were not significant (p < 0.05) in rats exposed to 2.5 and 5% DFPS and sperm counts were not changed in the 2.5% DFPS animals (Figure 4). Furthermore, treatment with FPS supplemented diet at 2.5, 5 and 10% resulted in mild elevation of abnormal sperm morphology in comparison with control animals. This increase was more prominent in 5 and 10% DFPS treated rats (Figure 4). The major sperm defects consisted of head abnormalities, midpiece, and tail abnormalities including curved midpiece, bent midpiece, looped tail, bent tail, headless sperms, and tailless sperms. However, 60 days DFPS post-treatment, the abnormal sperm morphology reduced while the sperm count, motile sperm and viability improved significantly (p < 0.05) compared to the corresponding initial treatment values (Figure 4).

Figure 4. Effects of dietary fluted pumpkin seeds (DFPS) on sperm quality variables of male rats after 60 days treatment and 60 days post-treatment. Cauda epididymis excised from each rats were used to evaluate sperm quality parameters as described in the material and methods section. (A) % motile sperms, (B) sperm count, (C) % abnormal sperm morphology, and (D) % sperm viability. Data are presented as the mean ± SD (n = 5). *versus control group, **versus 2.5% DFPS group, ***versus 5% DFPS group, and ****versus 10% DFPS group (p < 0.05).

There were dose-dependent marked alterations in the testicular cyto-architecture of rats fed DFPS. Histological examination of the control testes showed normal architecture with no visible lesion and intact seminiferous tubules (Figure 5). In contrast, morphological aberrations were seen in the histological features of rats exposed to fluted pumpkin seed diet especially in the 10% DFPS animals were severe loss of epithelial germ cells (spermatogonia, spermatocytes and spermatids) and Sertoli cells were observed. Disorganized seminiferous epithelium, defoliation of cells into the lumen, mild loss of epithelial germ cells and degeneration of the seminiferous epithelium were most of the histological features found in the tubules of animals exposed to the 2.5% and 5% DFPS (Figure 5).

Figure 5. Representative photomicrographs of the testes of experimental animals. (A) Control: No visible lesion seen. (B) Representative testis section of rats fed 2.5% dietary fluted pumpkin seeds (DFPS) shows abnormal round spermatids (starred), expanded lumen (Lu). (C) Representative testis section of rats in 2.5% DFPS (REV) group showing normal seminiferous tubules with normal spermatids (starred) and other germ cells (Spg: spermatogonia; Spct: spermatocytes) at different level of development. (D) Representative testis section of rats fed 5% DFPS shows disorganized seminiferous epithelium (starred), degenerated epithelium (Se), undefined lumen (Lu), detached epithelium (De). (E) Representative testis section of rats in 5% DFPS (REV) group shows seminiferous tubules with normal spermatogenesis (Lu: Lumen, Spg: spermatogonia, Spct: spermatocytes, Rs: round spermatids, Spz: spermatozoa, Int: interstitial areas. (F & G) Representative testis section of rats fed 10% DFPS shows severe loss of epithelial germ cells, tubular germ cell depletion, empty tubules without epithelial germ cells, distended interstitial areas (Int), lumen (Lu), St: seminiferous tubules, Se: seminiferous epithelium. (H) Representative testis section of rats in 10% DFPS (REV) shows seminiferous tubules with evidence of normal spermatogenesis and intact seminiferous epithelium. Int: Interstitial areas, Lu: Lumen; Spg: spermatogonia; Spct: spermatocytes; Rs: round spermatids. H & E; Mag. ×400, 10% DFPS (F): ×100.

The histologic features of the testes 60 days after withdrawal of the test diet were similar to the control with no visible lesion and normal seminiferous tubules showing most of the germ cells at all stages of development (Figure 5). Furthermore, quantitative analyses of testes showed a marked reduction in seminiferous epithelial height, tubular diameter as well as marked elevation in percentage tubular lumen, which was more prominent in the 10% DFPS group compared to untreated rats. The administration of 10% DFPS resulted in the marked reduction of percentage tubular compartment, tubular length, absolute volume of tubular compartment and cross-sectional area of seminiferous tubules as well as elevation of percentage inter-tubular compartment, absolute volume of tubular lumen, absolute volume of inter-tubular compartment compared to control testis. Exposure to DFPS at 2.5% marginally increased the tubular compartment, tubular length, tubular lumen, but reduced the absolute volume of tubular compartment, absolute volume of inter-tubular compartment, cross-sectional area of seminiferous tubules, inter-tubular compartment, seminiferous epithelial height and tubular diameter. In the 5% DFPS group, the tubular diameter, tubular lumen, inter-tubular compartment and tubular length were marginally increased. 60 days after withdrawal of test diet, the testicular cyto-architecture looked similar to the control and the histologic features in the seminiferous tubules induced by DFPS treatments were reversed to near control (Tables 1–3).

Table 2. Morphometry of the tubular and intertubular compartment of the testis of adult rats treated with fluted pumpkin seeds supplemented diet for 60 days and 60 days after withdrawal of treatment.

Parameters	Tubular compartment (%)	Intertubular compartment (%)	AV of tubular lumen (ml)	Tubular length per gram of testis
Control	84.21 ± 3.54	15.79 ± 3.54	0.45 ± 0.08	11.70 ± 1.93
2.5% DFPS	86.34 ± 5.03	13.65 ± 5.03	0.42 ± 0.07	12.70 ± 3.47
5% DFPS	80.04 ± 4.97	19.96 ± 4.97	0.65 ± 0.10	13.95 ± 3.04
10% DFPS	47.87 ± 3.44*	52.12 ± 3.44*	1.74 ± 0.24*	8.78 ± 2.07*
2.5%DFPS (REV)	87.08 ± 4.22	12.92 ± 4.21	0.32 ± 0.08	18.16 ± 6.46
5%DFPS (REV)	86.83 ± 2.80	13.17 ± 2.80	0.34 ± 0.13	16.78 ± 6.85
10%DFPS (REV)	86.46 ± 1.26**	13.54 ± 1.26**	0.37 ± 0.11**	14.29 ± 3.33**

Each value represents the mean ± SD. *Significantly different from the control. **Significantly different from 10% DFPS (p < 0.05) (n = 5). A_V = absolute volume.

Table 3. Absolute volume of the tubular and intertubular compartments and cross-sectional area of the seminiferous tubules of the testis of adult rats treated with fluted pumpkin seeds supplemented diet for 60 days and 60 days after withdrawal of treatment.

Parameters	AV of tubular compartment (ml)	AV of intertubular compartment (ml)	AC of ST (×10^3 μm^2)
Control	2.0 ± 0.084	0.38 ± 0.08	12.61 ± 4.23
2.5% DFPS	1.73 ± 0.10	0.27 ± 0.10	10.31 ± 4.77
5% DFPS	1.75 ± 0.11	0.44 ± 0.11	14.50 ± 4.91
10% DFPS	1.08 ± 0.08*	1.08 ± 0.08*	6.14 ± 2.32*
2.5%DFPS (REV)	1.67 ± 0.08	0.25 ± 0.08	17.78 ± 4.30
5%DFPS (REV)	1.70 ± 0.05	0.26 ± 0.05	16.13 ± 5.28
10%DFPS (REV)	1.88 ± 0.03**	0.29 ± 0.03**	14.95 ± 5.27**

Each value represents the mean ± SD. *Significantly different from the control. **Significantly different from 10% DFPS (p < 0.05) (n = 5). ST = seminiferous tubules. A_V = absolute volume. A_C = cross-sectional area.

Gas chromatography-mass spectroscopy (GC-MS) analyses of dietary fluted pumpkin seed

Figure 6 highlights peaks of available phyto-constituents in the ethanolic extract of cooked pumpkin seed revealed by GC-MS analysis. Table 4 depicts the constituent compounds identified from the National Institute Standard and Technology (NIST) database and their concentration (area %) and retention time. The phyto-constituents identified include 2H-3,9a-methano-1-benzoxepin (11.62%), ethyl 14-methyl-hexadecanoate (11.10%), 17-octadecynoic acid (10.94%), (E)-9-octadecenoic acid ethyl ester (9.39%), 13-oxabicyclo [9.3.1] pentadecane (7.78%), 9,12,15-octadecatrienoic acid (6.27%), 2,6,10,14,18-pentamethyl-2,6,10,14,18-eicosapentaene (6.09%), 2-myristynoyl-glycinamide (5.65%). 9,12-octadecadienoic acid (5.35), n-hexadecanoic acid (3.15%) and heptadecafluorononanoic acid (2.31%).

Table 4. Chemical constituents of boiled fluted pumpkin seeds identified in the present study by gas chromatography and mass spectroscopy (GC-MS).

Peak	Compound	Retention time (min)	Area % number
1.	2,3-Butanediol	3.709	2.63
2.	2,3-Butanediol	3.767	1.87
3.	2-Butanol, 3-methyl-	3.977	0.37
4.	Urea, N,N-dimethyl	5.174	0.12
5.	Glycerin	5.415	1.18
6.	Malic acid	6.342	0.15
7.	Cyclohexanebutanal, 2-methyl-3-oxo-, cis-	11.306	0.55
8.	3,7,11,15-Tetramethyl-2-hexadecen-1-ol	15.496	0.64
9.	2,6,10,14,18-Pentamethyl-2,6,10,14,18-eicosapentaene	16.308	6.09
10.	Cyclopropanebutanoic acid	16.502	0.51
11.	n-Hexadecanoic acid	16.876	3.15
12.	Hexadecanoic acid, ethyl ester	16.876	3.15
13.	Cyclopropaneoctanoic acid	17.701	1.64
14.	7-Hexadecenoic acid	17.746	2.14
15.	Heptadecafluorononanoic acid, nonyl ester	17.927	2.31
16.	9,12-Octadecadienoic acid	18.200	5.35
17.	(E)-9-Octadecenoic acid ethyl ester	18.241	9.39
18.	Ethyl 14-methyl-hexadecanoate	18.416	11.10
19.	17-Octadecynoic acid	18.906	10.94
20.	9,12,15-Octadecatrienoic acid	19.238	6.27
21.	2H-3,9a-Methano-1-benzoxepin, octahydro	20.142	11.62
22.	13-Oxabicyclo[9.3.1]pentadecane, 15-chlor	20.490	7.78
23.	2-Myristynoyl-glycinamide	21.529	5.65
24.	2-Myristynoyl-glycinamide	21.529	5.65

The 24 phytoconstituents were characterized and identified on comparison of mass spectra of the constituents with the National Institute Standard and Technology (NIST).

Figure 6. Gas chromatography-mass spectroscopy (GC-MS) chromatogram of ethanol extract of dietary fluted pumpkin seeds (DFPS). Peaks in chromatogram correspond to different phyto-constituents in the fluted pumpkin seeds. The unknown spectrum pattern generated by the GC-MS analysis of ethanol extract of DFPS was compared with the spectra of known constituents in National Institute Standard and Technology (NIST) database.

Discussion

The sequence of spermatogenesis and reproductive viability can be influenced by plant bioactive compounds, dietary pattern and nutritional factors (Chauhan and Agarwal 2010; D’Cruz et al. 2010; Abarikwu et al. 2017). The reversibility of antispermatogenic effects of fluted pumpkin seeds (FPS) were illustrated in this study. The statistically unchanged final body, absolute and relative weight of the testes rats during administration of DFPS depicts absence of systemic toxicity (Verma and Singh 2017; Njoku et al. 2018). It is also important to note that unchanged testicular weight as a result of treatment with antifertility/antispermatogenic agents has been previously described by us and others (Mishra and Singh 2005; Thakur et al. 2014; Ain et al. 2018; Njoku et al. 2018). The decline in sperm viability, motility and epididymal sperm numbers as well as elevated abnormal sperm morphology in rats fed fluted pumpkin seed-supplemented diet compared to control values implies that DFPS adversely altered the quality of the stored sperms in the epididymis (Rajalakshmi 1992; Verma and Singh 2017). Indeed, a person’s fertility condition can be ascertained by estimating the sperm motility and sperm number as important markers of semen quality (Ghosh et al. 2015; Verma and Singh 2017; Ain et al. 2018). We had previously reported that DFPS altered sperm quality variables at doses higher than currently used in this study (Njoku et al. 2018). This confirms that plant derived products have the capacity to impair testicular functions by interfering with androgen synthesis and spermatogenesis (D’Cruz et al. 2010). Steroidogenesis together with the development of germ cells into mature spermatozoa are prime events critical for maintaining male fertility. These processes are controlled by the precise synthesis and release of FSH from the pituitary-gonadal axis (Naik et al. 2016). Fluctuations in the concentration of serum and intratesticular testosterone due to DFPS confirm their adverse effects on androgen production (Ghosh et al. 2015). Elevation/and or diminution in testosterone levels have been associated with aberration in semen quality (Meistrich and Shetty 2003; Abarikwu et al. 2017). Elevated testosterone has been posited to promote germ cell apoptosis via suppression of differentiation of germ cells while reduced testosterone is associated with diminished sperm quality variables (Meistrich and Shetty 2003; Abarikwu et al. 2012). Because the testosterone level was lower in the 5% and 10% DFPS treated animals, the alterations in sperm variables were more severe in these group of animals compared to animals fed 2.5% DFPS. It is plausible to adduce that DFPS exerts adverse effects on the reproductive process through multiple mechanisms. However, after 60 days post-withdrawal of DFPS, the serum and intratesticular testosterone concentrations reverted to near control values and was associated with improved sperm quality. Additionally, intratesticular FSH significantly recovered beyond control values in the 10%DFPS animals following withdrawal of fluted pumpkin seeds-supplemented diet.

Steroidogenic enzymes (3β-HSD and 17β-HSD), Sertoli cell marker, γ-GT and indexes of oxidative stress including thiobarbituric acid reactive substances, were also assessed to further ascertain the mechanisms through which DFPS impacted testicular function of rats. Consumption of 5% and 10% DFPS resulted in a marked increase in testicular γ-GT activity possibly depicting an interference on Sertoli cell maturation (Domínguez-Vías et al. 2017). Increase in testicular γ-GT activity induced by anti-fertility agents have been reported previously (Yakubu et al. 2008; Abarikwu et al. 2017). However, the elevated γ-GT in the 5% DFPS group reverted to near control values, the testicular γ-GT activity showed no recovery in the 10% DFPS (REV) post-withdrawal. The steroidogenic enzymes, 3β-HSD and 17β-HSD exert regulatory influence on the steroidogenesis cascade (Abarikwu et al. 2012). The decrease in the activity of 17β-HSD caused by DFPS treatment for 60 days agrees with earlier findings that plants with antifertility potential diminishes testicular 17β-HSD activity (Verma and Singh 2017). It was suggested that decrease in the activities of these enzymes leads to reduced concentration of blood testosterone thereby inhibiting testicular androgenesis (Jana et al. 2010). The decrease in the testosterone levels of the male rats following DFPS could also be due to an indirect effect on the hypothalamus that is responsible for the secretion and release of androgens (Abdel-Magied et al. 2001). The DFPS- mediated reduction of testosterone and FSH could imply that FPS has anti-androgenic activity via interference with some committed enzymes in the testosterone biosynthetic pathway. Another possibility is that DFPS might have inhibitory effects on Leydig cell function. Therefore, DFPS administration for 60 days in adult male rats induces testicular dysfunction in the animals by interfering with the androgen synthesis machinery (Saalu et al. 2010; Adisa et al. 2014; Njoku et al. 2018). However, the reversal in the level of testosterone to the control values following post- treatment of DFPS suggest that FPS might fit as a contraceptive agent.

The increase in thiobarbituric acid reactive substances e.g., MDA, in testes of DFPS treated animals might also have altered the integrity of steroidogenic apparatus in the Leydig cells making it vulnerable to oxidative damage (Diemer et al. 2003; Verma and Singh 2017). The increase in MDA level alongside decrease and/or increase in antioxidant defense machinery of many tissues entail the onset of oxidative stress (Abarikwu et al. 2018). It was observed that DFPS promoted MDA formation and altered the profiles of some antioxidant markers in the testis, including catalase and reduced GSH. This is understandable as the presence of reactive oxygen species production system and abundance of polyunsaturated fatty acids in the testes make it vulnerable to oxidative stress (Aitken and Roman 2008). The elevated MDA concentration in DFPS treated animals at all doses used in the present study reflects increased lipid peroxidation in the testis. This was accompanied with a decrease in GSH levels in the testes of 2.5 and 5% DFPS treated animals indicating that the antioxidant defense of the testis was already compromised (Abarikwu et al. 2018). It is important to note that impaired sperm quality together with testicular oxidative damage due to intake of fluted pumpkin leaves and seeds have been reported previously (Saalu et al. 2010; Adisa et al. 2014; Njoku et al. 2018). We think that the significant increase in catalase activity and GSH concentration in 10% DFPS animals is an attempt by the antioxidant defense of the testis to counter-attack increased oxidative stress and to limit tissue damage (Nelson et al. 2006; Abarikwu 2014). The reversal of this oxidative stress status to near control values following withdrawal of treatment indicates recovery from testicular oxidative damage.

Histological examination of the testes and morphometric analyses showed that DFPS damaged the cyto-architecture of the testis. This was most pronounced in the 10% DFPS animals leading to tubules with severely depleted epithelial germ cells, few spermatocytes and absent of spermatids and Sertoli cells in basement membrane of the germinal epithelium. Furthermore, the testis of the 10% DFPS animals showed diminished tubular diameter, epithelial height and tubular and inter-tubular compartments as well as cross sectional area of the tubules. Similar alterations in testicular architecture accompanied with low sperm quality upon exposure to plant extract with anti-fertility effects have also been reported (Ain et al. 2018). This is possibly a result of the cytotoxic effects of phyto-constituents in the fluted pumpkin seeds (Hu et al. 2018). However, withdrawal of DFPS led to a near complete reversal of the testicular histo-architecture toward normalcy.

Characterizations of the ethanolic extract of cooked fluted pumpkin seeds by GC-MS identified compounds such as 2H-3,9a-methano-1-benzoxepin (11.62%), ethyl 14-methyl-hexadecanoate (11.10%), 17-octadecynoic acid (10.94%), (E)-9-octadecenoic acid ethyl ester (9.39%), 13-oxabicyclo[9.3.1] pentadecane (7.78%), 9,12,15-octadecatrienoic acid (6.27%), 2,6,10,14,18-pentamethyl-2,6,10,14,18-eicosapentaene (6.09%), 2-myristynoyl-glycinamide (5.65%). 9,12-octadecadienoic acid (5.35), n-hexadecanoic acid (3.15%), heptadecafluorononanoic acid (2.31%) among others listed in Table 4. Of these compounds, n-hexadecanoic acid and 9,12-octadecadienoic are reported to have inhibitory effects on testicular androgen synthesis by inhibiting the activities of steroidogenic enzymes e.g., 5α-reductase (Upgade and Bhaskar 2013). Sertoli cells provide nutritive factors and molecules that are essential for the completion of spermatogenesis, but are highly vulnerable to extraneous damage (Lohiya et al. 2002). Hu et al. (2018) posited that hexadecanoic acid decreased sperm count by inducing the apoptosis of Sertoli cells. Thus, the suppressed sperm viability, maturational arrest of germ cells (e.g., spermatocytes and spermatids) observed in DFPS treated rats is partly due to the damaging effects of constituent saturated fatty acids on Sertoli cells. Additionally, Sertoli cells that are not supported by adequate intra-testicular FSH stimulation might lead to germ cell apoptosis (Sofikitis et al. 2008; Shaha et al. 2010). The inadequate FSH levels in the 10% DFPS animals which does not sufficiently support Sertoli cells functions might also be the reason why most of the tubules in these animals have few Sertoli cells and nearly empty tubular compartments. It is therefore speculated in the present study that DFPS could directly kill Sertoli cells to arrest spermatogenesis. It is therefore thought that the observed oligospermia could be due to selective action of FPS on developing germ cells, possibly mediated via Sertoli cells. Furthermore, severe oligospermia (concentrations of less than 1 million sperm per/mL) is thought to decrease the chances of conception to less than 1% per year, and has emerge as a reasonable goal for male contraceptive research (Amory 2016).

The available evidence shows that a FPS-supplemented diet promotes spermatogenic arrest in adult rat’s testes through multiple mechanisms including direct killing of Sertoli cells. The reversal of the antifertility effects of FPS on testicular functions makes it a potential male contraceptive candidate. However, further studies to confirm this view are therefore warranted.

Material and methods

Preparation of dietary fluted pumpkin seed (DFPS)-supplemented diet

Seeds were obtained from mature fluted pumpkin (Telfairia occidentalis Hook) fruits sourced locally in Rivers State, Nigeria. These seeds were identified and certified by Dr. Ekeke Chimezia of the Department of Plant Science and Biotechnology, University of Port Harcourt, with a specimen number of UPH/P/096. The obtained fluted FPS were washed and boiled for 1 h at 100^°C. The boiled fleshy kernels of the seeds were sun-dried, ground and used to supplement standard rat feed. The test diet containing 2.5% (25 g FPS/kg), 5% (50 g FPS/kg) and 10% (100 g FPS/kg) of the boiled and ground seeds and 97.5%, 95% and 90% standard feed respectively, was mixed thoroughly and made into pellets. The formulated diet was sun-dried and used as test chow. Sun dried pelletized standard feed was used as control diet. Furthermore, part of the pulverized cooked kernel of the FPS were extracted with absolute ethanol and used for gas chromatography-mass spectroscopy (GC-MS) analysis.

Animals and treatment

Healthy male Wistar rats of average body weight 135 g were used for this experiment. These rats were sheltered in well aerated plastic cages for a 12-h dark/light interval and given standard rat feed and water ad libitum. Prior to treatment period, rats were allowed to acclimatize for one week. The handling of the animals and experimental procedures were in accordance with the ethical standards of the National Institute of Health Guidelines for Animal Care and Use of Laboratory Animals (Publication Number, 85–23) and were approved by Institutional Research Ethics Committee at the University of Port Harcourt. After acclimatization, rats were divided into seven groups of five animals each:

Control: Rats were fed standard pelletized chow

2.5% DFPS: Rats were fed 2.5% FPS-supplemented diet for 60 days

5% DFPS: Rats were fed 5% FPS-supplemented diet for 60 days

10% DFPS: Rats were fed with 10% FPS-supplemented diet for 60 days

2.5% DFPS (REV): 60 days of post-withdrawal of 2.5% DFPS supplemented diet

5% DFPS (REV): 60 days of post-withdrawal of 5% DFPS supplemented diet

10% DFPS (REV): 60 days of post-withdrawal of 10% DFPS supplemented diet.

After the treatment period, rats were euthanized with diethyl ether and serum was obtained for hormonal assays. Testes from each rat were removed and the left testis was blended (1:9) in ice-cold phosphate buffered saline (0.01M, pH = 7.4), and centrifuged at 5000 g at 4°C for 5 min to obtain the homogenate used for the biochemical assays. The right testis was fixed in Bouin’s solution and processed for histopathology and testicular morphometry. Cauda epididymides were used to evaluate sperm quality parameters.

Biochemical assay and procedures

Lipid peroxidation was evaluated by measurement of malondialdeyde (MDA) concentrations in the samples at 532 nm during the reactions of thiobarbituric and MDA as described by Ohkawa et al. (1979). The concentration of MDA values were expressed as micromolar of MDA per gram of tissue.

Glutathione levels were measured in the testicular homogenates according to the method of Moron et al. (1979) using the Ellman’s reagent, 5, 5ʹ-dithiobis-2-nitrobenzoic as the substrate. The concentration of GSH measured was expressed as unit per milligram of protein. Protein concentrations were determined as described by Lowry et al. (1951). Bovine serum albumin was used as standard.

Catalase activity in the testes was monitored according the procedure detailed by Clairborne (1995) with slight modifications. Briefly, 1 mL of the testicular homogenate was added to 2.5 mL of phosphate buffer (50 mM, pH 7.4) together with 0.4 mL H₂O₂ (60 mM). The change in absorbance was monitored at 240 nm against a blank for 3 min at 30 s interval which indicated the decomposition of H₂O₂. Catalase activities were calculated using the extinction coefficient of H₂O₂, 43.59 L/mol cm. One unit of catalase activity equals the amount of protein that converts 1 mmol H₂O₂ per minute.

The activity of the steroidogenic enzymes, 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-HSD were evaluated in the testis homogenate as described previously (Abarikwu et al. 2012). One unit of the enzyme activity is the amount causing a change in absorbance of 0.001 min⁻¹ at 340 nm.

Testicular γ-glutamyl transferase (γ-GT) activity was estimated as outlined by Szasz (1969). Briefly, 0.1 mL of supernatant was added to a 2.9 mL of a solution mixture of glycylglycine (0.16M) and L-γ-glutamyl-3-carboxy-4-nitroanilide in Tris base buffer (0.005M, pH:8.2) containing 0.016M magnesium chloride and the absorbance was recorded at 405 nm at 60 s intervals for 5 min.

Estimation of follicle-stimulating hormone and testosterone concentrations

Serum- and intratesticular-free FSH concentrations were quantified using a rat specific FSH enzyme linked immunosorbent assay kit (Elabscience Biotechnology Inc. Hubei, China). The absorbance was measured at 450 nm. The sensitivity of the FSH assay is 1.88 ng/mL. The inter- and intra-assay precision coefficient of variation was 5.05 and 4.36 respectively. Free testosterone level in both serum and testes homogenates (ITT) were quantified using AccuBind^TM testosterone kit (Monobind Inc. CA, USA) in accordance to the manufacturer’s instructions. The absorbance was recorded at 450 nm. The minimum detection limit in the testosterone assay was 0.1 ng/ml. The cross-reactivity assay with other steroids tested showed 0.01%.

Estimation of sperm motility, viability and morphology

Cauda epididymis crushed in normal saline and sieved with a nylon mesh was used to obtain the epididymal sperm numbers. Neubauer hemacytometer was used to count the spermatozoa according to the method illustrated by Abarikwu et al. (2015). Motile sperms sequestered from the epididymis were estimated visually at 400× magnification as described by Abarikwu et al. (2010). Portions of sperm concentrate dropped on a slide glass was smeared out with another slide, fixed in 95% ethanol, and stained with 1% eosin and 5% nigrosine was used for viability and morphological estimation. Deformity in various areas of spermatozoa was estimated according to the procedure illustrated by Abarikwu et al. (2010).

Histopathology and testicular morphometry

Testes fixed in Bouin’s solution for 24 h were dehydrated in ethanol grades and the organs subsequently embedded in paraffin wax. Five-μm of the tissue was severed, placed on slides and stained with hematoxylin and eosin for assessment. The height of the seminiferous epithelium and diameter of the seminiferous tubules were determined in all stages of the cycles from numerous images as described previously (Ribeiro et al. 2014). More so, the tubular diameter, seminiferous tubule epithelial height, volumetric proportions of seminiferous tubule, tubular lumen and interstitium were also measured with little modification were the Image J software, a 91 point grid was superimposed over images at × 400 magnification. To overlay the grid, the area of the image was first measured using the ‘measure’ tool of the Image J software, and each testicular structure touched by the grid point including seminiferous tubules; tubular lumen and interstitum were quantified with the aid of the ‘cell counter’ tool of Image J and expressed as percentage of the analyzed field. Length of the seminiferous tubules per testis was ascertained with the formular TL = STV/R2 (STV = seminiferous tubule volume; R = tubular radio) while cross sectional area of the seminiferous tubules was determined πR2 (where π is equivalent to 3.142 and R is the mean tubular radio of the seminiferous tubules). Absolute volume of the testis was also calculated with the formular: Av of testicular structure analyzed = (volume density (%) of structure/100 × testicular volume) while testicular volume was considered equal to testicular weight (França et al. 2000).

Gas chromatography-mass spectroscopy analysis

To perform GC-MS analysis, 1–2 μL of test sample was injected into the column of Shimadzu QP2010 Gas-Chromatography–Mass spectroscopy (GC-MS QP2010SE) at injector temperature of 280ºC and the split ratio of 30:1 The constituent compounds were separated using a fused silica column packed with Elite −5MS (5% Phenyl 95% dimethylpolysiloxane, 30 m × 250 μm). The oven temperature was elevated from 60°C to 250°C at 13°C/min for final hold of 4 min. The carrier gas, helium (99.99%), was passed through the column at constant flow rate of 1 mL/min. The constituent compounds corresponding to different spectrum were compared with the spectrum of known components in the National Institute Standard and Technology database. The name, molecular weight and structure of the components of the test materials were ascertained.

Statistical analysis

Statistical evaluation of obtained parameters was conducted with SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA) and data denoted as mean ± SD. All the data were analyzed by ANOVA followed by Tukey’s post-hoc test. The values of p < 0.05 were considered to indicate statistical significance.

Authors’ contributions

Supervised the study, designed the study, and approved the final version of the manuscript for submission: SOA; Participated in the supervision of study: AAU; Prepared the diet, performed the experiment, and wrote the draft version manuscript: RCN; Participated in performing the experiment: CYE, CJM.

Acknowledgment

The effort of the technical staff in the Postgraduate Research Laboratory of the Department of Biochemistry toward the completion of this study is appreciated.

Disclosure statement

No potential conflict of interest was reported by the authors.