Histopathological and Immunohistochemical Evaluation of Methotrexate-Induced Gonadal Damage in Rats: Role of SCF, mTOR, and SIRT-1

Abstract

Objective: Methotrexate (MTX) is a highly effective chemotherapy for cancer. This drug has a gonadotoxic effect, mainly in the testes and ovaries. Our study used histopathological and immunohistochemical methods to assess the potential damage to testicular and ovarian tissue caused by MTX use.

Methods: Twenty-four Wistar albino rats, both male and female, were used in our study. Four sets of rats; control male, MTX male, control female, and MTX female were created. The male and female MTX-treated groups received a single intraperitoneal dose of 20 mg/kg MTX. The testes and ovaries of rats sacrificed under general anesthesia were extracted and histopathologically analyzed. In addition, the immunoreactivity intensities of stem cell factor (SCF), mechanistic target of rapamycin (mTOR), and SIRT-1 in both tissues were measured by immunohistochemistry.

Results: Johnsen's testicular biopsy score in the testicular seminiferous tubules was significantly lower in the MTX group than in the control group (p<0.001). The ovary showed substantial follicular degeneration (p<0.05), vascular congestion (p<0.01), and fibrosis (p<0.001). MTX reduced SCF immunoreactivity density in the testis and ovary (p<0.05). Furthermore, MTX reduced mTOR, a marker of autophagy, in the testis (p<0.05) and ovary (p<0.001) compared with the control. SIRT-1 intensity increased dramatically in the testis (p<0.001) and ovary (p<0.01) in the injured group, unlike the mTOR marker.

Conclusions: Our investigation revealed that the gonads incurred significant damage as a result of MTX. One vital option for reducing or eliminating this damage to the ovaries and testicles is the use of anti-oxidant-rich substances.

Keywords: Methotrexate; chemotherapy; ovary; testis.

Figure 1

Light microscopic findings and JBTS of rat testis tissue. Control male group; ST: Seminiferous tubules. MTX Male group; N: Necrotic seminiferous tubules; orange arrow: descending spermatogenic lineage cells; blue arrow: tubular vacuolization; green triangle: Asymmetrical seminiferous tubule morphology. H&E: hematoxylin-eosin and MT staining (Olympus BX51, Tokyo, Japan. X20). JBTS graph of the experimental groups. Data are presented as mean ± standard deviation or median (min-max). *: p<0.05, **: p<0.01, ***: p<0.001 JBTS: Johnsen’s testicular biopsy score, MTX: Methotrexate

Figure 2

Light microscopic findings of the rat ovary tissue. Control Female group: Several developmental phases of folliculi are observed. Primordial follicles (*), primary follicles (PF), secondary follicles (SF), and Corpora lutea (CL) with large, weakly pigmented acidophilic cells, mature Graafian follicles (GF). MTX Female group: Degenerative follicles (DF). Green arrow, vascular congestion; red star, fibrosis. H&E: hematoxylin-eosin and Masson’s trichrome staining (Olympus BX51, Tokyo, Japan. X20). MTX: Methotrexate, H&E: Hematoxylin&Eosin.

Figure 3

Histopathological findings of rat ovary tissue. Graph exhibiting degenerative follicles, vascular congestion, and fibrosis in the experimental groups. Data are presented as mean±standard deviation or median (min-max). *: p<0.05, **: p<0.01, ***: p<0.001. MTX: Methotrexate, Min-max: Minimum-maximum.

Figure 4

Immunohistochemical microscopic findings and graphs of SCF markers in testicular and ovarian tissues from the experimental groups. The brown areas indicate immunostaining. The slides were counterstained with hematoxylin. Data are presented as mean±standard deviation or median (min-max).  *: p<0.05, **: p<0.01, ***: p<0.001. (Olympus BX51, Tokyo, Japan. X20). SCF: Stem cell factor, Min-max: Minimum-maximum, MTX: Methotrexate.

Figure 5

Immunohistochemical microscopic findings and graphs of mTOR markers in testicular and ovarian tissues from the experimental groups. The brown areas indicate immunostaining. The slides were counterstained with hematoxylin. Data are presented as mean±standard deviation or median (min-max).  *: p<0.05, **: p<0.01, ***: p<0.001. (Olympus BX51, Tokyo, Japan. X20). MTX: Methotrexate, mTOR: Mechanistic target of rapamycin, Min-max: Minimum-maximum.

Figure 6

Immunohistochemical microscopic findings and graphs of SIRT-1 markers in testicular and ovarian tissues from the experimental groups. The brown areas indicate immunostaining. The slides were counterstained with hematoxylin. Data are presented as mean±standard deviation or median (min-max).  *: p<0.05, **: p<0.01, ***: p<0.001. (Olympus BX51, Tokyo, Japan. X20). Min-max: Minimum-maximum, MTX: Methotrexate.