The antineoplastic busulphan impairs peritubular and Leydig cells, and vitamin B12 stimulates spermatogonia proliferation and prevents busulphan-induced germ cell death

Abstract

Busulphan (Bu), an alkylating agent used for bone marrow and spermatogonial stem cell transplantation (SSCT), impairs Sertoli (SC) cells, which are necessary for the spermatogonial stem cell (SSC) homing during transplantation. As Leydig (LC) and peritubular myoid (PMC) cells are essential for SC support and maintenance of spermatogonial niche, we evaluated the impact of Bu on the LC and PMC structural integrity. Vitamin B (B) has demonstrated beneficial effects against drug-induced testicular changes; thus, we also examined whether this vitamin is able to stimulate spermatogonia mitotic activity and prevent Bu-induced germ cell death. Rats received 10mg/kg of Bu in the 1st and 4th days, and daily B supplementation during Bu treatment and for 6days after the last injection of Bu (Bu-6d), totaling 10days of treatment. Other animals received the same treatment as Bu-6d, and B supplementation (Bu+7dB) or saline (Bu+7dS) for 7 more days, totaling 17days of treatment. Serum testosterone levels were measured. In the historesin-embedded testis sections, the seminiferous tubule and epithelial areas were measured, and the number of spermatogonia and PMC was quantified. Actin and 17β-HSD6 immunofluorescence was detected, and the number of TUNEL-positive LC and germ cells was computed. In Bu-6d, PMC number reduced, and a weak actin immunoexpression and death in these cells was observed. The testosterone levels reduced, and the interstitial tissue showed a weak 17β-HSD6 immunoexpression and increased number of TUNEL-positive LC. In Bu+7dB, the number of spermatogonia was higher than in Bu-6d and Bu+7dS, and the number of TUNEL-positive germ cells was significantly lower than in Bu+7dS. Bu exerts a harmful impact on PMC and LC, reducing the testosterone levels. Vitamin B prevents significantly Bu-induced germ cell death and stimulates spermatogonia proliferation, being a useful strategy for the enrichment of SSC in vitro and an adjuvant therapy for spermatogenesis recovery in oncologic patients.

Vitamin B12 Prevents Cimetidine-Induced Androgenic Failure and Damage to Sperm Quality in Rats

“…In previous studies, we have demonstrated beneficial effects of vitamin B 12 on the sperm concentration and spermatogenesis of cimetidine-treated rats (11, 12). A similar effect has also been demonstrated in the seminiferous epithelium of animals receiving co-administration of the antineoplastic busulphan and vitamin B 12 (22, 23). However, a preventive effect of vitamin on the sperm exposed to these drugs has not yet been reported.…”

“…Vitamin B 12 exerts a potential role in spermatogenesis (12, 21–23), and sperm parameters in humans (61) and animals (12, 20, 21, 62), and has been a therapeutic agent used for the treatment of male infertility, especially oligozoospermia or asthenozoospermia (63). In previous studies, we have demonstrated beneficial effects of vitamin B 12 on the sperm concentration and spermatogenesis of cimetidine-treated rats (11, 12).…”

“…The sections were incubated in 2% BSA to block non-specific binding and washed in phosphate-buffered saline (PBS; pH 7.4). The slides were incubated overnight in a humidified chamber at 4°C with the following primary antibodies: rabbit anti-activated caspase-3 antibody (Abcam; Cambridge, UK; ab-4051), diluted 1:100 (7); and rabbit anti-17β-HSD6 antibody (Santa Cruz Biotechnology Inc., Dallas, USA; sc-101878), at a concentration of 2 μg/ml, according to Beltrame et al (7) and Sasso-Cerri et al (23). The sections incubated with anti-activated caspase-3 antibody were washed in PBS and incubated at room temperature with Labeled StreptAvidin-Biotin Kit (Universal Dako LSAB, Dako Inc., Carpinteria, CA, USA), and the reaction was revealed with 3,3′-diaminobenzidine (DAB, Sigma-Aldrich, USA).…”

“…Vitamin B 12 is able to recover the seminiferous epithelium of animals treated with cimetidine (11), increasing the number of spermatogonia and spermatocytes, and ameliorating the sperm concentration (12). A potential effect of vitamin B 12 on the spermatogenic recovery has also been demonstrated in animals receiving co-administration of the antineoplastic busulphan and vitamin B 12 (22, 23).…”

Exposure to Chemotherapy During Childhood or Adulthood and Consequences on Spermatogenesis and Male Fertility

“…No study investigated the impact of anticancer treatment on macrophages, despite their role as "guardians of fertility" [83]. To the best of our knowledge, only Sasso-Cerri et al have shown a nuclear fragmentation and a depletion of peritubular myoid cells in adult rat testes after alkylating agent exposure [84].…”