Seminiferous epithelium damage after short period of busulphan treatment in adult rats and vitamin B₁₂ efficacy in the recovery of spermatogonial germ cells

Summary

Several different strategies have been adopted in attempt to recover from chemotherapy‐damaged spermatogenesis that is often seen in oncologic patients. In this study, we have evaluated the impact of short period of exposure to busulphan on the haemogram and seminiferous epithelium of adult rats, focusing on spermatogonial depletion and Sertoli cell (SC) integrity. We then examined whether vitamin B₁₂ supplementation improves the haematological parameters and spermatogonia number. The animals received 10 mg/kg of busulphan (BuG) or busulfan+vitamin B₁₂ (Bu/B₁₂G) on the first and fourth days of treatment. In H.E.‐stained testicular sections, the areas of the seminiferous tubule (ST) and seminiferous epithelium were measured. The number of spermatogonia in H.E‐stained and PCNA‐immunolabelled testicular sections was quantified. The frequency of tubules with abnormal SC nuclei or TUNEL‐positive SC was evaluated. Vimentin immunofluorescence in ST was also evaluated. In BuG and Bu/B₁₂G, the animals showed leukopenia and thrombocytopenia, but the body weight reduced only in BuG. The areas of ST and seminiferous epithelium decreased in Bu/B₁₂G and BuG. In BuG, the number of H.E.‐stained and PCNA‐immunolabelled spermatogonia reduced significantly. The frequency of tubules containing abnormal SC nuclei and TUNEL‐positive SC increased and the vimentin immunoexpression pattern changed. In Bu/B₁₂G, the number of H.E.‐stained or PCNA‐immunolabelled spermatogonia increased fourfold in comparison with BuG. The structural changes in ST after 6 days of busulphan exposure may be associated with the potential effect of this anti‐neoplastic agent on SC. The increased number of spermatogonia in the busulphan‐treated animals receiving vitamin B₁₂ indicates that this vitamin can be an adjuvant therapy to improve the fertility in male cancer patients.

Introduction

Busulphan (1,4‐butanediol dimethanesulphonate) is an alkylating agent that causes cytotoxicity due to its capacity to transfer alkyl groups to cellular constituents (Buggia et al. 1994; Iwamoto et al. 2004), forming cross‐links in DNA intra‐ or interstrands and DNA–protein links (Sanderson & Shield 1996). These linkages caused by alkylation drastically alter the cell replication, DNA repair and gene transcription (Galaup & Paci 2013). Therefore, busulphan has been a useful agent for the treatment of haematological diseases, such as chronic myelogenous leukaemia (Buggia et al. 1994; Galaup & Paci 2013), and has also been used before haematopoietic stem cell transplantation (HSCT) due to its toxic effect on these stem cells (Buggia et al. 1994; Meng et al. 2003). The action of this alkylating agent on haematopoiesis leads to thrombocytopenic effect and reduction in the number of leucocytes (Gibson et al. 2003; Molyneux et al. 2011). Although alkylating chemotherapeutic agents have been used routinely for conditioning prior to HSCT, these protocols have been associated with high risk of infertility in the oncological patients (Anserini et al. 2002; Green et al. 2010; Wilhelmsson et al. 2014). Fifty per cent of the patients conditioned with cyclophosphamide plus busulphan, for example, remain azoospermic (Anserini et al. 2002).

In male rodents, busulphan has been demonstrated to cause sterility due to death of spermatogonial cells (van Keulen & de Rooij 1974; Bucci & Meistrich 1987; Anjamrooz et al. 2007; Benavides‐Garcia et al. 2015). In male rats treated with busulphan at different doses (13–40 mg/kg), the seminiferous tubules showed depletion of most spermatogonia and some spermatocytes (de Rooij & Kramer 1970; Bucci & Meistrich 1987). In rodents, an increase in the incidence of apoptotic spermatogonia has been observed after busulphan treatment (Choi et al. 2004). Thus, due to the potential depletion effect of busulphan on the spermatogonial germ cells, this drug has been widely used to prepare recipients for spermatogonial stem cell (SSC) transplantation in different species (Wang et al. 2010; Qu et al. 2012; Qin et al. 2015).

Numerous studies have been performed in attempt to find strategies to recover chemotherapy‐damaged spermatogenesis and treat infertility in oncologic patients (Grigg et al. 2000; Anserini et al. 2002; Benavides‐Garcia et al. 2015). Semen freezing and intracytoplasmic sperm injection (ICSI) have been clinically applied in adults (Clark et al. 2011). Moreover, the strategies to recover spermatogenesis after SSC transplantation have been tested in different species and have demonstrated to be clinically applied to reestablish gamete production and fertility in patients with cancer (Geens et al. 2008; Clark et al. 2011; Benavides‐Garcia et al. 2015). However, ICSI cannot be applied in prepubertal boys who are not yet producing sperm (Benavides‐Garcia et al. 2015), and due to the small testicular biopsies from young patients, in vitro SSC enrichment is necessary for clinical SSC transplantation (Geens et al. 2008; Clark et al. 2011).

Vitamin B₁₂, also known as cobalamin, has been widely used as therapeutic agent for the treatment of male infertility (Watson 1962; Iwasaki et al. 2003; Chatterjee et al. 2006). This vitamin is a water‐soluble vitamin that plays an important role in DNA synthesis and cell division (Oh & Brown 2003) and is an essential cofactor for methionine synthase during the production of purines and pyrimidines (O'Leary & Samman 2010), being essential for the processes that require high cell renewal rate. Patients with lung cancer who received cisplatin/pemetrexed chemotherapy and were supplemented with vitamin B₁₂ showed a significant reduction in the haematologic toxicity, mainly in neutropenia (Vogelzang et al. 2003).

Testicular alterations such as reduction in testis weight, atrophy of seminiferous tubules, spermatogenesis arrest and spermatid as well as sperm aplasia have been demonstrated in vitamin B₁₂‐deficient rodents; however, the supplementation of the animals with vitamin B₁₂ has been shown to recover the testicular functions (Kawata et al. 2004; Watanabe et al. 2007). Spermatogenic improvement has also been demonstrated when a large amount of vitamin B₁₂ is given to patients with oligospermia (Sharp & Witts 1962). Moreover, this vitamin has been a therapeutic agent for the treatment of male infertility (Iwasaki et al. 2003; Chatterjee et al. 2006). In rodents, vitamin B₁₂ is able to recover the doxorubicin‐induced testicular damage (Ozaki et al. 1988; Oshio et al. 1989) and stimulates spermatogenesis in cimetidine‐treated rats, avoiding tubular atrophy (Beltrame et al. 2011). Therefore, vitamin B₁₂ supplementation seems to be a useful alternative therapy for spermatogenesis recovery.

After busulphan‐based conditioning for HSCT in childhood, the recovery of spermatogenesis is a slow progressive process (Wilhelmsson et al. 2014). In the majority of adult men who received this alkylating agent, some detectable sperm can only be found 5 years after transplantation (Grigg et al. 2000). Therefore, studies focusing on adjuvant therapies to improve the male fertility in patients with cancer are necessary. Numerous studies have focused on the busulphan‐induced spermatogonial injury; however, the effect of this alkylating agent on the structure of Sertoli cells (SC) is scarce in the literature. In this study, we evaluated the impact of short period of exposure to low dose of busulphan in the haemogram and seminiferous epithelium of adult rats, focusing on the spermatogonia number and the structure of Sertoli cells. We also verified whether vitamin B₁₂ supplementation is able to improve the haematological parameters and recover the spermatogonial germ cells.

Material and methods

Animals and treatment

Adult Holtzman male rats aged 100 days were maintained in polypropylene cages under 12‐h light/12‐h dark cycle at controlled temperature (23 ± 2°C) and humidity (55 ± 10%), with water and food provided ad libitum. The animal care was conducted following the national law on animal use (CONCEA, Brasília, DF, Brazil).

Twenty‐five animals were distributed into five groups, containing five animals each: busulphan (BuG), vehicle (VG), control (CG), busulphan/vitamin B₁₂ (Bu/B₁₂G) and vitamin B₁₂ (B₁₂G) groups (Figure 1). The animals from BuG group received two intraperitoneal injections of 10 mg of busulphan per kg bw, diluted in polyethylene glycol (Zarbock et al. 2006). The doses of busulphan+polyethylene glycol and polyethylene glycol only were applied in the first and fourth days of treatment to the animals from BuG and VG group respectively (Figure 1). In all other days of treatment, the animals from these groups received only saline. The animals from Bu/B₁₂G and B₁₂G received 3 μg of vitamin B₁₂‐cyanocobalamin (Bedozil^®, 5000 μg – Bunker Indústria Farmacêutica LTDA, São Paulo, SP, Brazil). This dose of vitamin was determined from a preliminary evaluation of the quantity of daily food intake by the animals at this age (Beltrame et al. 2011). Thus, the animals from Bu/B₁₂G and B₁₂G received intraperitoneal injections of busulphan+polyethylene glycol+vitamin B₁₂ and vitamin B₁₂+polyethylene glycol, respectively, in the first and fourth days of treatment. These animals received vitamin B₁₂ in all other days of treatment. The animals from CG received saline in all days of treatment (Figure 1).

Figure 1.

Treatment schedule applied to the animals from CG, BuG, Bu/B₁₂G and B₁₂G.

Blood collection for haemogram and Histological procedures

After the treatment (day 11), the animals were weighed and anaesthetized with 80 mg/Kg bw of ketamine hydrochloride (Francotar; Virbac do Brasil Ind. Com. Ltda., Jurubatuba, São Paulo, Brazil) and 7 mg/Kg bw of xylazine hydrochloride (Virbaxyl; Virbac do Brasil Ind. Com. Ltda). The blood samples were collected from the ventricles in tubes containing 3.2% buffered sodium citrate solution (BD Vacutainer^® Citrate Tubes; BD Biosciences, Curitiba, Paraná, Brazil). The tubes were mixed according to the manufacturer's recommendation and the blood was analysed immediately. The white blood cell (WBC) and platelet (PLT) counts were determined on Sysmex XS‐1000i^™ Automated Hematology Analyzer (Sysmex do Brasil Ind Com Ltda, São José dos Pinhais, Paraná, Brazil) at São Lucas Clinical and Microbiological Laboratory (Araraquara, SP, Brazil).

Testes were removed and weighed, and the testicular fragments were fixed for 48 h in 4% formaldehyde freshly prepared from paraformaldehyde (Merck, Darmstadt, Germany) buffered at pH 7.4 with 0.1 M sodium phosphate. Subsequently, the testicular fragments were dehydrated in graded ethanol and embedded in glycol methacrylate (historesin); the testicular fragments were dehydrated, diaphanized and embedded in paraffin. Historesin sections were stained with H.E. or picrosirius (Cerri & Sasso‐Cerri 2003) for morphometric and morphological analyses, while the paraffin sections were subjected to immunofluorescence analysis.

Morphometrical analysis

The following morphometrical analyses were performed in three H.E.‐stained non‐serial testicular sections per animal. In each section, 15 tubules were randomly chosen, totalling 45 tubules per animal. The images were captured by a camera (DP‐71; Olympus, Tokyo, Japan) attached to a light microscope (BX‐51; Olympus).

Seminiferous tubule areas

The area of the seminiferous tubule section, the area of seminiferous epithelium and the luminal area were measured using an image analysis system (Image Pro‐Express 6.0; Olympus). Only round cross‐sections of tubules showing a defined tubular lumen (filled or not by detached germ cells) were measured.

Number of spermatogonia in H.E.‐stained sections

In the H.E.‐stained testicular sections, spermatogonial cells were identified according to Dym and Clermont (1970). In 45 seminiferous tubule cross‐sections per animal, the number of spermatogonia was quantified, and the number of spermatogonia per tubule and per μm² of seminiferous tubule was calculated.

TUNEL method

The protocol of TUNEL method was performed according to the Apoptag^® Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore; Temecula, CA, USA) and as previously described (Sasso‐Cerri & Cerri 2008; Beltrame et al. 2011).

Frequency of tubules containing abnormal SC nuclei and TUNEL‐positive SC

The frequency of tubules containing abnormal SC nuclei was determined in 45 H.E.‐stained tubules per animal. The following SC nuclear features were considered: irregular nuclear shape, enhanced basophilia, condensed masses of chromatin, the presence of halos/vacuoles as well as the presence of dislocated nuclei from the basal compartment to the luminal portion of the seminiferous tubule. The frequency of tubules in which SC nuclei exhibited weak nucleolar staining was also examined. Among the sections subjected to TUNEL method, 45 tubular sections were also examined and the frequency of tubules containing TUNEL‐positive SC was calculated.

Immunofluorescence for the detection of vimentin and proliferating cellular nuclear antigen (PCNA)

Paraffin‐embedded testicular sections were adhered to silanized slides and immersed in 0.001M sodium citrate buffer pH 6.0 and maintained at 90°C in a microwave oven for the antigen recovery. After the inactivation of endogenous peroxidase, the slides were washed in Tris–phosphate‐buffered saline (TBS) pH 7.2 and incubated with 2% Bovine serum albumin (BSA). The testicular sections were incubated in a humidified chamber at 4°C overnight with the following primary antibodies: 1) mouse anti‐vimentin monoclonal antibody (V9; Sigma‐Aldrich, St. Louis, MO, USA), diluted 1:30; 2) mouse anti‐PCNA monoclonal antibody (Biocare Medical; Concord CA, USA), diluted 1:100. The sections were washed in TBS and incubated with secondary anti‐mouse antibody (Alexa Fluor 488^®; Molecular Probes by Life Technologies; Carlsbad, CA, USA) for 1 h at room temperature. The nuclear staining was performed with DAPI (Molecular Probes by Life Technologies; Carlsbad, CA, USA). Negative controls were incubated in non‐immune serum instead of primary antibodies. The sections were analysed using a fluorescent microscope DM400 B LED, a camera DFC‐550 and an Image Analysis System LAS4 (Leica Microsystems, Wetzlar, Germany).

Number of PCNA‐immunolabelled spermatogonia

Among the sections subjected to PCNA immunofluorescence, the number of PCNA‐immunolabelled spermatogonia was quantified in 45 seminiferous tubules per animal.

Statistical analyses

All data are presented as mean±SD, and the statistical analysis was performed using the GraphPad Prism^® 6.0 software (GraphPad Software, CA, USA). The results were submitted to the one‐way anova followed by Tukey's post‐test. The significance level considered was P ˂ 0.05.

Ethical approval statement

The protocol of this study was approved by the Ethical Committee on Animal Use of Dental School, São Paulo State University – FOAr/UNESP, Brazil (CEUA no. 03/2011).

Results

Haemogram

A significant reduction in the number of white blood cells and platelets was observed in the animals from BuG and Bu/B₁₂G in comparison with the other groups. No significant difference was found between CG, VG and B₁₂G (Table 1).

Table 1.

Number of white blood cells (WBC) and platelets (PLT)

Groups	WBC (103/mm3)	PLT (103/mm3)
CG	5.7 ± 2.1a	691 ± 59a
VG	5.6 ± 1.9a	650 ± 67a
B12G	5.5 ± 2.4a	626 ± 68a
BuG	1.2 ± 0.4b	220 ± 91b
Bu/B12G	1.4 ± 0.6b	229 ± 90b

a ≠ b (P ˂ 0.05).

Body and testicular weight

As shown in Table 2, the body weight of the animals from BuG reduced significantly (around 20%) in comparison with CG, VG and B₁₂G. However, in the animals treated with busulphan and supplemented with vitamin (Bu/B₁₂G), the body weight was found to be similar to those in CG, VG and B₁₂G.

Table 2.

Body and testicular weights

Groups	Body weight (g)	Testes weight (g)
CG	330.0 ± 33.9a	1.67 ± 0.11a
VG	340.4 ± 11.3a	1.54 ± 0.10a
B12G	340.6 ± 21.7a	1.55 ± 0.05a
BuG	266.0 ± 19.9b	1.53 ± 0.06a
Bu/B12G	339.0 ± 14.8a	1.60 ± 0.12a

a ≠ b (P ˂ 0.05).

Histopathological features

In the testicular sections of animals from CG, VG and B₁₂G, the seminiferous tubule sections showed normal features, including the preserved histological integrity of epithelium and the absent or scarce sloughed germ cells in the tubular lumen (Figure 2a,b and 3a,c,e). In both BuG and Bu/B₁₂G, the seminiferous tubules were reduced (Figure 2c,d), and intraepithelial vacuoles and detached germ cells in the tubular lumen were found, mainly in BuG (Figure 2c). In the last group, spermatocytes with abnormal nuclear features and the depletion of spermatogonia were observed in the seminiferous epithelium (Figure 3g). However, in Bu/B₁₂G, scarce spermatogonia were observed in the tubules (Figure 3l). In these busulphan‐treated groups, germ cells (probably spermatogonia) exhibiting strong basophilic condensed masses of chromatin in the nuclear periphery, typical of apoptosis, were found (Figure 3h,m). The occurrence of apoptosis was confirmed by the presence of TUNEL‐positive spermatogonia (Figure 4a,b). Sertoli cell nuclei with evident apoptotic features such as irregular nuclear shape, intense and homogeneous basophilia and condensed portions of chromatin in the nuclear periphery were also observed in the busulphan‐treated groups (Figure 3i–k,n–p). In these groups, TUNEL‐positive SC was also found, confirming the occurrence of cell death in these cells (Figure 4c–f).

Figure 2.

(a–d) Photomicrographs of the testicular sections of animals from CG (a), B₁₂G (b), BuG (c) and Bu/B₁₂G (d) stained by H.E. In a and b, the seminiferous tubule sections show normal epithelial histoarchitecture. In c and d, the tubular sections are smaller than in a and b. In c, intraepithelial vacuoles (arrows) and detached germ cells in the tubular lumen (asterisks) are observed. Bars = 59 μm.

Figure 3.

(a–p) Photomicrographs of the seminiferous tubule sections of the animals from CG (a,b), VG (c,d), B₁₂G (e,f), BuG (g–k) and Bu/B₁₂G (l–p) stained by H.E. In CG (a,b) and B₁₂G (e,f), spermatogonia (arrows) and Sertoli cell nuclei (Sc) are normally observed in the epithelial basal compartment. In b and d, the Sertoli cell nuclei (Sc) show typical nucleolus (arrowheads) with satellite karyosomes. In f, spermatogonia in mitosis (arrows). In BuG (g), note the depletion of spermatogonia (asterisks) and numerous spermatocytes with condensed chromatin, suggesting apoptosis (arrows). The Sertoli cells show weak nucleolar staining (Sc). In h, germ cell with clumps of condensed chromatin in the nuclear periphery – typical of apoptosis (arrows). In i, the Sertoli cell shows irregular and strongly basophilic nucleus. In j and k, abnormal Sertoli cell nuclei show masses of condensed chromatin in the nuclear periphery, typical of apoptosis (arrows). In l (Bu/B₁₂G), Sertoli cell nuclei (Sc) and spermatogonia (arrows) are observed. In m, a germ cell with clumps of condensed chromatin (arrows), typical of apoptosis, is observed in close contact with a Sertoli cell nucleus (Sc). In n–p, Sertoli cell nuclei show condensed masses of chromatin in the periphery (arrows), typical of apoptosis; satellite karyosomes are observed (arrowheads). Sp (spermatocytes); St (round spermatids); Sz (elongate spermatids). Bars = 14 μm (a,c,e,f,g and l); 6 μm (b,d,h–k,n–p); 7 μm (m).

Figure 4.

(a–f) Photomicrographs of the seminiferous tubule sections of BuG (a,c and d) and Bu/B₁₂G (b,e and f) subjected to TUNEL method. TUNEL‐positive spermatogonia (a and b) and TUNEL‐positive Sertoli cells (c–f) are observed (arrows). Bars = 6 μm.

Effect of busulphan on the frequency of tubules containing abnormal SC and TUNEL‐positive SC

The frequency of H.E.‐stained tubules containing abnormal SC nuclei increased significantly (65%) in BuG when compared to CG group. Moreover, the SC nucleoli were weakly stained in BuG when compared to CG and VG (Figure 5a–c). Weak nucleolar staining was also observed in the sections stained by picrosirius (Figure 5d,e). A high frequency (80%) of tubules containing SC with weakly stained nucleoli was found in BuG. In this group, a significant increase in the frequency of tubules containing TUNEL‐positive SC (25%) was also observed (Figure 6).

Figure 5.

(a–e) Photomicrographs of the seminiferous tubule sections of CG (a and d), VG (b) and BuG (c and e) stained by H.E. (a–c) and picrosirius (d and e). When compared to a and b, the Sertoli cell nuclei in c show weak nucleolar staining (arrows). In d, strongly picrosirius‐stained nucleoli (arrowheads) are observed in the Sertoli cells, whereas in e, the SC nucleoli are weakly stained (arrowheads). Bars = 14 μm (a–c); 6 μm (d,e).

Figure 6.

Frequencies of the seminiferous tubules containing abnormal SC nuclei (SCN), TUNEL‐positive SC and SC with weak nucleolar staining (SCnu) in CG and BuG. *statistically significant (P ˂ 0.05).

Effect of busulphan on the vimentin immunoexpression

In CG, vimentin immunoexpression was observed surrounding SC nuclei and extending throughout the cytoplasm to the apical portion (Figure 7a–c). However, changes in the pattern of vimentin immunoexpression were observed in BuG. In this group, thick and strong immunofluorescent masses of vimentin were located in the basal portion of the tubules, indicating collapse of these filaments (Figure 7d–f). In the sections used as negative control, immunofluorescent structures were not found (data not shown).

Figure 7.

(a–f) Photomicrographs of the seminiferous tubule sections subjected to immunofluorescence for detection of vimentin in CG (a–c) and BuG (d–f). In b and c, thin vimentin‐immunofluorescent extensions are observed from the basal towards to the luminal portion of the tubule. In e and f, thick masses of immunofluorescent vimentin can be observed in the basal portion of the tubule in BuG (arrows). Bars = 14 μm.

Effect of Bu and Bu/B₁₂ on the areas of seminiferous tubule, seminiferous epithelium and lumen

In the testes of the animals from BuG and Bu/B₁₂G groups, the areas of seminiferous tubule and seminiferous epithelium decreased significantly (P  ˂ 0.05) in comparison with the other groups. No difference in the luminal area was found between the groups (Table 3).

Table 3.

Areas of seminiferous tubule (TA), seminiferous epithelium (EA) and lumen (LA)

Groups	TA (μm2)	EA (μm2)	LA (μm2)
CG	87819 ± 2166a	78313 ± 3611a	9506 ± 2135a
VG	86178 ± 4634a	78290 ± 4018a	7887 ± 1280a
B12G	84956 ± 3861a	77149 ± 3798a	7807 ± 1558a
BuG	78581 ± 1263b	69884 ± 1457b	8697 ± 1530a
Bu/B12G	78706 ± 1706b	70929 ± 1248b	7776 ± 1317a

a ≠ b: statistically significant (P ˂ 0.05).

Effect of Bu and Bu/B₁₂ on the number of spermatogonia and PCNA immunoexpression

No statistically significant difference was observed in the number of spermatogonia/tubule or the number of spermatogonia/μm² of seminiferous tubules between CG, VG and B₁₂G. On the other hand, a statistically significant reduction in the number of these cells was observed in BuG (95%) and Bu/B₁₂G (77%) when compared to CG (Table 4). However, in Bu/B₁₂G, the number of these cells was significantly higher (fourfold) when compared with the animals treated with busulphan alone (Table 4).

Table 4.

Number of spermatogonia per tubule and number of spermatogonia per μm² of seminiferous tubules

Groups	Spermatogonia/tubule	Spermatogonia/μm2 (×10−3)
CG	11 ± 0.8a	6.3 × 10−3 ± 0.6 × 10−3 a
VG	11 ± 1.7a	6.5 × 10−3 ± 0.8 × 10−3 a
B12G	11 ± 0.9a	6.4 × 10−3 ± 0.5 × 10−3 a
BuG	0.5 ± 0.2b	0.4 × 10−3 ± 0.1 × 10−3 b
Bu/B12G	2.5 ± 0.7c	1.5 × 10−3 ± 0.4 × 10−3 c

a ≠ b ≠ c: statistically significant (P ˂ 0.05).

Numerous PCNA‐immunolabelled spermatogonia were observed in the seminiferous epithelium of animals from CG and VG (Figure 8a–f; Figure 9). In BuG, scarce or no immunolabelled spermatogonia were observed in the seminiferous tubules (Figure 8g–i; Figure 9), while in Bu/B₁₂G, few PCNA‐immunolabelled spermatogonia were found in the tubules (Figure 8j–l; Figure 9). In the sections used as negative control, immunofluorescent structures were not observed (data not shown).

Figure 8.

(a–l) Photomicrographs of the seminiferous tubule sections of CG (a–c), VG (d–f), BuG (g–i) and Bu/B₁₂G (j–l) subjected to immunofluorescence for the detection of PCNA. In b,c,e,f,k and l, PCNA‐immunofluorescent spermatogonia (arrows) are observed in the basal compartment of the seminiferous epithelium, while in BuG (h and i), no immunolabelled spermatogonia is found. Bars = 6 μm.

Figure 9.

Number of PCNA‐immunolabelled spermatogonia in CG, BuG and Bu/B₁₂G groups. a$\ne$b$\ne$c: statistically significant (p<0.05).

Discussion

Our findings showed that after only six days of the last injection of busulphan (totalling 20 mg/kg b.w.), a significant reduction in the body weight as well as leukopenia and thrombocytopenia was observed in the busulphan‐treated adult rats (BuG). In the testes, the treatment caused a reduction in the area of the seminiferous epithelium and, despite the short period of treatment, a massive loss of spermatogonia and SC structural changes were evident. Although the supplementation with vitamin B₁₂ was not able to improve the haemogram, the body weight was similar to that of the control animals after the treatment, and the number of spermatogonia increased fourfold in the busulphan‐treated rats supplemented with vitamin B₁₂ (Bu/B₁₂G).

Reduction in body weight has also been demonstrated in busulphan‐treated rodents (Jiang 1998; Gibson et al. 2003). In the present study, the supplementation of the busulphan‐treated animals with vitamin B₁₂ was able to avoid weight loss, probably due to its effect on the appetite. Therapy with vitamin B₁₂ in patients with tropical sprue resulted in the prompt return of appetite, increased dietary intake and weight gain (Klipstein & Corcino 1977). Although the body weight was maintained in the animals from Bu/B₁₂ group, leukopenia and thrombocytopenia were evident in this group, similar to BuG. In mice, either the number of white blood cells or that of platelets decreased after one day of the treatment with 10.5 mg/kg of busulphan (Gibson et al. 2003). Busulphan induces hypocellular bone marrow (aplastic anaemia) due to damage to haematopoietic stem cells (HSCs) (Gibson et al. 2003; Meng et al. 2003). Therefore, this drug has been used to deplete HSCs in patients who need bone marrow transplantation (Buggia et al. 1994; Meng et al. 2003). Based on these data and despite the potential effect of vitamin B₁₂ on DNA synthesis and cell division (Oh & Brown 2003), if busulphan treatment caused a myeloablative effect in the animals, an improvement in the haemogram by the supplementation with vitamin B₁₂ would not be expected after few days of treatment.

In busulphan‐treated rodents, a reduction in the testes weight has been observed after 3–6 weeks of treatment and correlated with a massive loss of germ cells (Gomes et al. 1973; Anjamrooz et al. 2007). In the present study, although the areas of the seminiferous tubule and seminiferous epithelium decreased significantly, the testes weight did not change in both busulphan‐treated groups (BuG and Bu/B₁₂G), indicating that the period by which the animals were exposed to busulphan (6 days after the last dose) was not sufficient to induce a massive loss of germ cells and a subsequent reduction in the testes weight. However, the animals that received busulphan alone showed sloughed germ cells in the tubular lumen and a significant reduction (95%) in the number of spermatogonia. These findings are in agreement with previous studies in which low doses of busulphan (10 mg/kg to 20 mg/kg) have caused a reduction in the number of spermatogonia (de Rooij & Kramer 1970; Bucci & Meistrich 1987; Haddad et al. 1997; Jiang 1998; Kopecky et al. 2005). In rodents, spermatogonial depletion has also been reported between 5 and 10 days after the treatment with 30 mg/kg (O'Shaughnessy et al. 2008) and 21 days after the treatment with 10–20 mg/kg (Kopecky et al. 2005) of busulphan. In rats after 11 days of treatment with doses of 13 mg/kg of busulphan, the testes showed depletion of pachytene spermatocytes, indicating a cytotoxic effect on the spermatogonial cells at the time of treatment. In the animals treated with 28 and 40 mg/kg of busulphan, the seminiferous tubules showed an absence of early spermatocytes and most spermatogonia, indicating a cytotoxic effect on the spermatogonial stem cells (Bucci & Meistrich 1987). Thus, when low doses of busulphan are administered, the remaining surviving stem cells are able to repopulate the seminiferous epithelium (Choi et al. 2006). In the present study, the treatment with busulphan was not able to exterminate the spermatogonial germ cells, and the vitamin supplementation probably stimulated the remaining spermatogonia because the number of these cells increased fourfold in Bu/B₁₂G when compared with the animals that received only busulphan.

Vitamin B₁₂ has been demonstrated to play an essential role in the maintenance of testicular functions in vitamin B₁₂‐deficient rodents (Kawata et al. 2004; Watanabe et al. 2007) and has been a therapeutic agent for the treatment of male infertility (Sharp & Witts 1962; Iwasaki et al. 2003; Chatterjee et al. 2006). This vitamin has also been able to recover the doxorubicin‐induced testicular damage (Ozaki et al. 1988; Oshio et al. 1989), stimulates spermatogenesis and avoids tubular atrophy in cimetidine‐treated rats (Beltrame et al. 2011). These effects may be related to the role of vitamin B₁₂ in the cellular proliferation because this vitamin is an essential cofactor for methionine synthase during the production of purines and pyrimidines (O'Leary & Samman 2010), stimulating DNA synthesis and cell division (Beck et al. 1965; Oh & Brown 2003). A high incidence of PCNA‐positive spermatogonia and spermatocytes has been shown in the testes of rats treated with histamine H₂ receptor antagonist – cimetidine – and supplemented with vitamin B₁₂ (Beltrame et al. 2011). Moreover, a detailed quantification of spermatogonial cell types in stage‐specific seminiferous tubules showed that the supplementation of rats with vitamin B₁₂ during cimetidine treatment for 52 days seems to stimulate specifically type A spermatogonia mitotic activity, which in turn recovers the number of spermatogonia and spermatocytes (Beltrame & Sasso‐Cerri 2016). In the present study, the number of PCNA‐immunolabelled spermatogonia was higher in the animals from Bu/B₁₂G in comparison with BuG, suggesting that vitamin B₁₂ was able to induce spermatogonial mitotic activity. However, it is also important to take into account that the vitamin supplementation could protect partially the spermatogonial cells, avoiding busulphan‐induced cell death in some of these cells.

It is important to emphasize that the number of spermatogonia of the animals that received only vitamin B₁₂ (B₁₂ group) was similar to that of the animals from vehicle and control groups. This result may be related to the fact that B complex vitamins are water‐soluble vitamins, and the excess of these vitamins is excreted in the urine and stored in liver when the organism is supplied (Andrès et al. 2004). Therefore, we could hypothesize that vitamin B₁₂ would not stimulate the spermatogonial mitotic activity in a nutritionally balanced seminiferous epithelium. Otherwise, the supplementation would be useful and effective under pathological conditions in which spermatogenic process is devoid of vitamin. This idea can be reinforced by the fact that patients treated with busulphan have shown decreased vitamin B₁₂ serum levels (Anger et al. 1979). Another important point that could explain why the number of spermatogonia did not increase in B₁₂G is the balance between apoptosis and cell proliferation when the tissue is in homoeostasis. In rats supplemented with vitamin B₁₂, numerous PCNA‐immunolabelled spermatogonia and early primary spermatocytes have been observed in parallel to a high incidence of TUNEL positivity in these same germ cells (Beltrame & Sasso‐Cerri 2016). As mitotic divisions of spermatogonia are synchronized to meiotic divisions of spermatocytes, the concomitant presence of apoptotic spermatogonia and apoptotic spermatocytes in the same tubule sections might also indicate that this synchronization is carried out through the synchronized control of mitotic and meiotic checkpoints (Blanco‐Rodríguez et al. 2003).

Changes in the seminiferous tubules such as the lack of the epithelial structural integrity (Kopecky et al. 2005; Pospechova et al. 2007) and reduction in the tubular diameter (Anjamrooz et al. 2007) have also been detected in mice after a long period of post‐treatment with 20 mg/kg of busulphan. In the present study, the reduction in the areas of seminiferous tubule and seminiferous epithelium confirms that a short period of treatment with busulphan is able to disturb the structural integrity of the seminiferous epithelium. In some histopathological disorders, a significant increase in the tubular lumen occurs due to the loss of germ cells (Blanco et al. 2007). Otherwise, when Sertoli cells (SC) are affected, the structural integrity of the basal compartment changes and the lack of germ cells are followed by tubular shrinkage without increasing the lumen (Hooley et al. 2009; Beltrame et al. 2011). Our results showed that although the epithelial area reduced significantly, the luminal area was unaltered, indicating that the loss of germ cells was accompanied by a seminiferous tubule shrinkage, which is confirmed by the reduction in the area of the seminiferous tubule. This result may be related to the harmful effect of busulphan on SC structural integrity, which was confirmed by the presence of SC nuclei with irregular nuclear shape, intense basophilia, condensed masses of chromatin in the nuclear periphery and TUNEL labelling, indicating apoptosis, as previously demonstrated in the SC of cimetidine‐damaged tubules (Sasso‐Cerri & Cerri 2008; Beltrame et al. 2011). Moreover, TUNEL‐positive SCs were found in 25% of tubules in BuG (i.e. after only six days of exposure to busulphan). It is not surprising that SC undergoes cell death by a cytotoxic alkylating agent such as busulphan. Alkylation of DNA induces apoptosis through the generation of hydrogen peroxide (Tada‐Oikawa et al. 1999). Besides nuclear changes, SC also showed different vimentin immunoexpression pattern in BuG. Vimentin, intermediate filament abundant in Sertoli cells (Franke et al. 1979), is centred around the nucleus and extends towards desmosome‐like junctions with early spermatogenic cells and into the apical cytoplasm (Amlani & Vogl 1988). As demonstrated in the present study, collapse of vimentin filaments around the SC nuclei has also been reported after some weeks of the last injection of busulphan (two doses of 10 mg/kg) in rats (Kopecky et al. 2005). Busulphan treatment has also been demonstrated to cause a reduction in the expression of P‐cadherin, which maintains SC–germ cell attachment, leading to the structural changes in the seminiferous epithelium of rats (Pospechova et al. 2007). Thus, the reduction in the epithelial and tubular areas observed after only six days of post‐treatment with busulphan can be directly related to the interference of the treatment in the structural integrity of Sertoli cells. However, the way by which this structural disarrangement starts in SC needs to be clarified. Intriguingly, most seminiferous tubules of the BuG animals showed Sertoli cell nuclei with weak nucleolar staining in both H.E.‐ and picrosirius‐stained sections. It is known that nucleolus is the place of ribosome biogenesis, and chemotherapeutic drugs such as 5‐fluorouracil (Burger et al. 2010) and triptolide (Leuenroth & Crews 2008) induce nucleolar damage and inhibit ribosome biogenesis. Thus, the ribosome biogenesis (in the nucleolus) has been considered the highest sensitive cellular process towards chemotherapeutic drugs (Burger et al. 2010). In BuG, the presence of SC nuclei with apoptotic features associated with the changes in the nucleolar staining pattern suggests that SC nucleolus is a possible target for busulphan. This idea is reinforced by the fact that the nucleolus is the main place controlling the stability and degradation of p53 pathway, which induces apoptosis (Burger et al. 2010). According to Rubbi and Milner (2003), a direct disarrangement of the nucleolar structure activates p53‐induced apoptosis. Therefore, understanding the effect of busulphan on the nuclear structure of SC would be useful for the elaboration of strategies to preserve busulphan‐depleted epithelium integrity and guarantee a more stable spermatogenesis after spermatogonial cell transplantation.

In conclusion, busulphan at low dose and short period of treatment induces thrombocytopenia, leukopenia and changes in the seminiferous tubules of adult rats. The presence of structural changes in SC after the short period of exposure to busulphan confirms that this cell seems to be a potential target of this anti‐neoplastic drug. The supplementation with vitamin B₁₂ avoids body weight loss and improves the number of spermatogonia due to its effect on the spermatogonial mitotic activity. These results indicate the potential efficacy of vitamin B₁₂ adjuvant therapy to improve the male fertility in patients with cancer.

Conflict of interest

All the authors declare no competing financial interests.