1 Asian journal of andrology 2009 Vol: 11(5):583-599. DOI: 10.1038/aja.2009.25

Sperm characteristics and ultrastructure of testes of rats after long-term treatment with the methanol subfraction of Carica papaya seeds.

The contraceptive efficacy of Carica papaya seeds after short-term evaluation has been well established. We have examined the safety and mechanism of contraception in rats after long-term treatment with the methanol subfraction (MSF) of C. papaya seeds. The test substance was administered orally to the male albino rats (n = 40) at 50 mg per kg body weight each day for 360 days. Control animals (n = 40) received olive oil as a vehicle. Recovery was assessed up to 120 days after treatment withdrawal. Sperm parameters, serum testosterone levels, fertility, histology and ultrastructure of the testis, haematology and serum clinical chemistry were evaluated to establish the safety and efficacy of the test substance. Safety of long-term treatment was evidenced by unaltered health status, organ weight, haematology and clinical chemistry, and by an increase in body weight. The mechanism of contraception was shown by reduction in nuclear and cytoplasmic volume, normal nuclear characteristics and vacuolization in the cytoplasmic organelles of the Sertoli cells, as well as nuclear degeneration in spermatocytes and spermatids indicating disturbed spermatogenesis. Leydig cells were normal. Initial effects were observed in Sertoli cells at 60 days of treatment. Spermatocytes and spermatids were affected after 120-240 days of treatment. A significant decline in sperm count and viability, total inhibition of sperm motility, increased numbers of sperm abnormalities, normal serum testosterone levels and 100% sterility were evident after 60 days of treatment. All the altered parameters, including percent fertility, were restored to control level 120 days after treatment withdrawal. It is concluded that the MSF is safe for long-term treatment and the mechanism of contraception is shown by its effect on spermatid differentiation in the testis, possibly mediated by the Sertoli cell factors.

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Figures
Figure 1: Histology of the testis of a control animal. The seminiferous tubules contain Sertoli cells and germ cells of various stages, covering the entire process of spermatogenesis. The lumen contains mature spermatozoa. The interstitium contains distinct Leydig cells. Scale bar = 100 m. S, Sertoli cells; SG, spermatogonia; SC, spermatocytes; R, round spermatid; E, elongated spermatid; SP, spermatozoa; LY, Leydig cells. Figure 2: Ultrastructure of the testis of control animals, showing a secondary spermatocyte. The nucleus contains condensed chromatin material and a distinct nuclear membrane. Mitochondria are scattered throughout the cytoplasm. Scale bar = 2.17 m. M, mitochondria; N, nucleus; NM, nuclear membrane. Figure 3: Ultrastructure of the testis of control animals showing a round spermatid. The nucleus is round, with a prominent nuclear membrane. Mitochondria occupy the periphery of the cytoplasm. Golgi vesicles are well defined. Scale bar = 2.78 m. GV, Golgi vesicles; N, nucleus; NM, nuclear membrane. Figure 4: Ultrastructure of the testis of a control animal, showing a Leydig cell. The cell is characterized by a round nucleus, containing a prominent nuclear membrane. The cytoplasm shows vesicles of smooth endoplasmic reticulum, prosecretory granules, prominent mitochondria and lipid bodies. Scale bar = 2.78 m. L, lipid bodies; NM, nuclear membrane; P, prosecretory granules; SER, smooth endoplasmic reticulum. Figure 5: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 120 days showing the Sertoli cells. Vacuolization in the cytoplasm is evident. Secretory granules, prosecretory granules, mitochondria and Golgi bodies are scattered in the cytoplasm. The nucleus appears normal, with indentations and patchy chromatin material. Association of germ cells with the cytoplasm of the Sertoli cells appears normal. Scale bar = 2.38 m. S, Sertoli cell; SP, spermatid; N, nucleus. Figure 6: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 120 days, showing the spermatocytes, with distinct nucleus, patchy chromatin material and normal cytoplasm. Cytoplasmic organelles, however, are poorly defined. The plasma membranes of the spermatocytes are distinct and show closer association with the Sertoli cell cytoplasm . Scale bar = 2.78 m. SC, spermatocytes; N, nucleus. Figure 7: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 120 days, showing the spermatids. The nucleus appears round, with well-defined nuclear membranes and chromatin structures. The cytoplasm shows scattered mitochondria and cisternae of rough endoplasmic reticulum. The acrosome vesicles are evidenced by accumulation of Golgi vesicles. Scale bar = 2.78 m. SP, spermatids; N, nucleus; M, mitochondria; GV, Golgi vesicles. Figure 8: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 240 days, showing the Sertoli cells. The nucleus shows indentations and patchy chromatin material. Mitochondria show vacuolization. The cytoplasm of the associated germ cells was poorly defined. Scale bar = 2.78 m. N, nucleus. Figure 9: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 240 days, showing the Sertoli cells. The nucleus shows deep indentations, with patchy chromatin material, and the cytoplasmic organelles are fewer in number. Mitochondria are sparse and vacuolated. Scale bar = 2.38 m. N, nucleus; M, mitochondria. Figure 10: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 240 days, showing the spermatocytes. The nucleus shows signs of pyknosis. Mitochondria show vacuolization. Scale bar = 2.38 m. N, Nucleus; M, mitochondria. Figure 11: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 240 days, showing a round spermatid. The nucleus shows signs of pyknosis. Scale bar = 2.38 m. N, nucleus. Figure 12: Histology of the testis of rats treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 60 days. Vacuolization in Sertoli cells is evident. Spermatogonia, spermatocytes and spermatids are unaffected, showing normal nuclear and cytoplasmic characteristics. The lumen contains spermatozoa. The Leydig cells are normal. Scale bar = 100 m. S, Sertoli cells; SG, spermatogonia; SC, spermatocytes; E, elongated spermatid; B, basal lamina; LY, Leydig cells; R, round spermatid; L, lumen. Figure 13: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 240 days, showing the elongated spermatids. The nuclear membranes are poorly defined (arrows). Mitochondria show vacuolization. Other cytoplasmic organelles are sparse. Scale bar = 2.78 m. Figure 14: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 360 days, showing the Sertoli cells. The nucleus is irregular, with deep indentations. Cytoplasmic organelles are relatively sparse. However, well-defined mitochondria are scattered throughout the cytoplasm. Scale bar = 2.17 m. M, mitochondria; N, nucleus; NM, nuclear membrane. Figure 15: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 360 days, showing the spermatocytes. The cell is severely affected. The nuclear membranes and nuclear contents are poorly defined. Cytoplasmic organelles are sparse. Scale bar = 2.38 m. N, nucleus; NM, nuclear membrane. Figure 16: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 360 days, showing the round spermatids. The nucleus shows signs of pyknosis. The cytoplasmic organelles are sparse. Scale bar = 2.78 m. N, nucleus; NM, nuclear membrane. Figure 17: Ultrastructure of the testis of rat treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 360 days, showing the Leydig cells. The cell appears normal. Lipid bodies, secretory granules, mitochondria and stacks of smooth endoplasmic reticulum are prominent. Nucleus and nuclear membrane appear normal. Scale bar = 2.78 m. L, lipid bodies; M, mitochondria; N, nucleus; NM, nuclear membrane; SER, smooth endoplasmic reticulum; SG, secretory granule. Figure 18: Histology of the testis of rats treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 120 days. Sertoli cells show vacuolization, whereas the spermatogonia, spermatocytes and spermatids (round as well as elongated) appear normal, with normal nuclear and cytoplasmic characteristics. A few of the spermatocytes show nuclear pyknosis (arrows). The cytoplasm of all germ cells appears granular. The lumen contains spermatozoa. The Leydig cells are normal. Scale bar = 100 m. S, Sertoli cells; SG, spermatogonia; SC, spermatocytes; R, round spermatid; E, elongated spermatid; SP, spermatozoa; LY, Leydig cells. Figure 19: Histology of the testis of rats treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 240 days. Disruption of spermatogenesis is evident. Vacuolization is evident in the cytoplasm of Sertoli cells, spermatocytes and spermatids. The nuclei of spermatocytes and spermatids show pyknosis. Spermatogonia and Leydig cells appear normal. The lumen contains spermatozoa. Scale bar = 100 m. S, Sertoli cells; SG, spermatogonia; SC, spermatocytes; R, round spermatid; E, elongated spermatid; SP, spermatozoa; LY, Leydig cells. Figure 20: Histology of the testis of rats treated with methanol subfraction (MSF) at 50 mg per kg body weight per day for 360 days. Degeneration of the epithelium is common in the majority of the seminiferous tubules. Sertoli cells show vacuolization, and the spermatocytes and spermatids show nuclear pyknosis and cytoplasmic vacuolization. The lumen contains germ cell debris and fewer spermatozoa. Leydig cells appear normal. Scale bar = 100 m. S, Sertoli cells; SG, spermatogonia; SC, spermatocytes; R, round spermatid; E, elongated spermatid; SP, spermatozoa; LY, Leydig cells. Figure 21: Histology of the testis of rats 120 days after treatment withdrawal. Restoration of spermatogenesis is evident. The seminiferous epithelium is comparable with that of a control animal. The Sertoli cells and germ cells show closer association to each other, with normal nuclear and cytoplasmic characteristics. The lumen contains packed spermatozoa, and the Leydig cells appear normal. Scale bar = 100 m. S, Sertoli cells; SG, spermatogonia; SC, spermatocytes; R, round spermatid; E, elongated spermatid; SP, spermatozoa; LY, Leydig cells. Figure 22: Ultrastructure of the testis of control animals showing the Sertoli cells. The nucleus appears irregular, with indentations. The cytoplasmic organelles indicate an active secretory state. The mitochondria, lipid droplets and rough endoplasmic reticulum are prominent and scattered throughout the cytoplasm. Secretory granules are abundant. Scale bar = 1.79 m. G, Golgi bodies; L, lipid droplets; M, mitochondria; N, nucleus; SG, secretory granules. Figure 23: Ultrastructure of the testis of control animals showing the Sertoli cell cytoplasm. The nucleus shows indentation and a prominent nucleolus. The mitochondria are well defined. Prosecretory granules, rosettes of glycogen granules and free ribosomes are scattered throughout the cytoplasm. Scale bar = 2 m. L, lipid droplets; M, mitochondria; N, nucleus; NU, nucleolus; P, prosecretory granules. Figure 24: Ultrastructure of the testis of control animals showing the primary spermatocytes. The cytoplasm appears granular. The nucleus contains distinct chromatin networks, with well-defined nucleoli and prominent nuclear membranes. Scale bar = 2.17 m. N, nucleus; NM, nuclear membrane.
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