Varicocele Time-dependently Affects DNA Integrity of Sperm Cells: Evidence for Lower In vitro Fertilization Rate in Varicocele-positive Rats

In this experimental study,twenty-four mature male Wistar rats, 10 weeks old that weighed 200 ± 14 g were used. The rats were purchased from the Animal Resources Center, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran and were acclimatized in an environmentally controlled room (temperature: 20-23°C and a 12 hours light/12hours dark schedule). Food and water were available ad libitum. In this study, all experiments conducted on the animals were in accordance with Urmia University’s guidance from the Ethical Committee for Research on Laboratory Animals. Following one week acclimation, the animals were assigned to four groups (n=6) of control-sham and test groups. The test group was further divided into 3 subgroups according to the study termination (4, 6 and 8 months) after varicocele induction.

In the test groups, the left varicocele was induced as previously reported (18). Briefly, following anesthesia with 5% ketamine (40 mg/kg, i.p.) and 2% xylazine (5 mg/kg, i.p.), the diameter of renal vein was reduced to 1 mm and a left renal vein ligation was performed medial to the junction of the adrenal and spermatic veins. Then, the anastomotic branches between the left testicular and left common iliac vein were ligated. The control-sham group, while under anesthesia, only underwent a simple laparatomy with no ligation.

Animals were euthanized on 4, 6 or 8 months following varicocele induction, by a special CO2 device. The testes were dissected out, fixed in 10% formalin fixative for histological investigations and subsequently embedded in paraffin. Sections (5-6 μm) were stained with Iron-Weigert (Pajohesh Asia, Iran) for detection of germinal cell nuclei in the testis. The sections were analyzed under light microscope at two magnifications (×400 and ×1000).

To estimate tubular differentiation index (TDI), the percentage of seminiferous tubules (STs) that contained more than three layers of differentiated germinal cells from spermatigonia type A were considered negative. Those which showed more than three and/or four layers were considered TDI positive. From each sample, 20 sections (6 μm) were prepared and analyzed.

We used the repopulation index (RI) to calculate the ratio of active spermatogonia (spermatogonia type B) with light nuclei to inactive spermatogonia (spermatogonia type A) with dark nuclei as seen by Iron-Weigert staining in STs from 20 prepared sections per sample, as described earlier (19, 20).

Epididymides were carefully separated from the testicles under x20 magnification with a stereo zoom microscope (Model TL2, Olympus Co., Tokyo, Japan). The epididymis was divided into 3 segments: head, body and tail. The epididymal tail was trimmed and minced in 5 mL Hams F10 medium. After 20 minutes, the ground epididymal tissue was separated from the released spermatozoa and counted. Smears were prepared by eosinnegrosin to evaluate dead, abnormal and morphologically immature sperm (MIS). Sperm which stained red were considered non-viable; those with cytoplasmic residuals were considered morphologically immature.

Aniline blue staining was performed in order to evaluate sperm nuclear maturity (or protamine deficiency). The proportion of motile spermatozoa was determined by counting 100 cells in randomly selected fields for each sample at 37°C (21).

The comet assay (Cat No. 1435723/34, Sigma Co., St. Louis, MO, USA) was used to investigate DNA fragmentation A ×40 objective device (Olympus, Germany) was linked to an epi-fluorescent microscope (Model GS7, Nikon Co., Japan) and used for visualization of DNA damage. Light green spots with typical tails were considered to be damaged DNA and spots with no tails were marked as normal DNA.

To evaluate DNA double strand breaks, an acridine orange staining kit (Sigma Co., St. Louis, MO, USA) was used. Samples were analyzed by epifluorescent microscope (Model GS7, Nikon Co., Japan). Sperm samples for acridine orange staining were provided as previously described (22).

Samples that contained spermatozoa were prepared from the sperm suspensions as mentioned earlier. The samples were incubated at 37°C under 5% CO2 (CO2 Incubator, LEEC, England) for 3 hours. Then, as previously described, 0.1 ml from superficial sperm and/or 0.1 ml from sediment sperm of suspensions in one tube were added to 150 μl of tissue culture medium (TCM) that contained the oocytes (23).

Mature female rats were injected subcutaneously with 7.5 IU pregnant mare’s serum hMG (hMG, Netherlands) 48 hours prior to an i.p. injection of 100 IU human chorionic gonadotropin hCG (Teikoku Zohki Co., korea). Rats were euthanized with a special CO2 device 24 hours after hCG injection. The oviducts were removed and the ampullar portion was placed into a plastic dish that contained PBS (pH=7.2). The oocytes that were in cumulus masses were dissected out of the oviducts and introduced into tissue culture medium 199 (TCM 199, Sigma Co., USA). A drop of medium with 2 oocytes was allocated with a 10 μl sperm suspension (total: 80,000 sperm). For each animal, a total of 20 oocytes were divided into 10 drops.

To determine the effect of varicocele on oxidative stress, total antioxidant capacity (TAOC) of the control-sham and test groups were measured. The assessment was performed based on the ferric reduction antioxidant power (FRAP) assay (19). Briefly, at low pH (acetate buffer, 300 mM, pH 3.6), reduction of the FeIII-TPTZ complex to the ferrous form produces an intensive blue color measurable at 593 nm. The intensity of the complex following addition of the appropriate volume of serum to the reducible solution of FeIII-TPTZ is directly related to the total reducing power of the electron donating antioxidant. An aqueous solution of FeII (FeSO4.7H2O) and appropriate concentration of freshly prepared ascorbic acid are used as blank and standard solutions, respectively.

The total sulfhydryl level in serum was measured according to Hu and Dillared method (19). In short, 0.2 ml serum was added to 0.6 ml Tris-EDTA buffer (Tris base 0.25 M, EDTA 20 mM, pH= 8.2) followed by the addition of 40 μl DTNB (10 mM in pure methanol) in a 10 ml glass test tube. The final volume of the mentioned mixture was made up to 4.0 ml by extra addition of methanol. After incubation for 15 minutes at room temperature, the samples were centrifuged at 3000 x g for 10 minutes and ultimately the absorbance of the supernatant was assessed at 412 nm.

Statistical analyses were performed on all numerical data by two-way ANOVA and SPSS software version 13.00. All values were expressed as mean ± SD. P<0.05 was considered statistically significant.

Light microscopic analyses of the seminiferous tubules (STs showed most tubules with germinal epithelium dissociation and disruption of the cellular junction between sertoli and spermatogenesis cells. More than 33.50 ± 5.80% of STs in the left testes and 21.25 ± 2.21% in the right testes .showed dissociated epithelium after 4 months of varicocele induction. This phenomenon increased over time; after 6 months, 41.50 ± 3.10% of STs, and at 8 months, 52.25 ± 1.70%, of STs demonstrated cellular dissociation in the left testes. The right testes of the test groups showed 34.25 ± 3.09% and 38.12 ± 1.93% dissociation in the germinal epithelium of tubules at 6 and 8 months after varicocele initiation, respectively. No histological alterations were noted in the control-sham group and the STs manifested with normal cellular junction (Fig 1).

Histological observations demonstrated that most STs had negative TDI, which further deteriorated with time in the test groups. The majority of STs showed depletion and reduction of germinal cell layers after 8 months. Right testes of the test animals showed relatively moderate destruction in STs when compared to the left testes. No histological abnormality was observed in the control-sham group (Fig 2).

Special nucleus staining (Iron-Weigert) was performed in order to differentiate inactive cells (spermatogonia type A) from active cells (spermatogonia type B). The results showed that after varicocele induction the RI was negative in most test group STs and the number of STs with negative RI increased over time in both the right and left testes. Although pathologic RI was present in both testes, the right testes of the test animals showed lower negative RI in comparison to the left (Fig 3-A and 3-B). Further analyses showed some STs with maturation arrest (MA) in the test groups that were substantiated by time. The quantitative data for TDI and MA are shown in table 1.

Hemocytometric evaluations for sperm count showed that after varicocele induction the sperm number reduced significantly (p<0.05); this reduction increased with time. Eight month varicoceleinduced cases manifested severely decreased sperm count; one of the six rats in this group was azoospermic. The right testes of varicocele-induced animals showed relatively higher sperm counts in all test groups when compared to the left testes. Aniline blue staining for sperm nucleus maturity revealed that the ratio of immature sperm with light stained nuclei increased remarkably in varicocele-induced animals (Fig 4A). The rats that suffered from varicocele for 8 months manifested higher numbers or percentages of immature sperm when compared with the other test groups. Furthermore, the sperm with cytoplasmic droplets were considered MIS. Observations demonstrated that the number of sperm with cytoplasmic droplets increased significantly (p<0.05) in varicocele-induced groups. Accordingly, 8 month varicocele-induced rats had the highest MIS value. In comparison to the left testes, the right testes showed a lower rate of immature sperm. A comparison of the results between sperm collected from the right testes of the varicocele-induced group with the control-sham group revealed that MIS was significantly (p<0.05) higher in right testes samples (Fig 4B).

Light microscopic analyses for sperm motility showed that the percentage motility decreased over time in varicocele-induced rats. Sperm delivered from the right testes of varicocelized animals exhibited relatively higher sperm motility in comparison to the left testes.

According to the results from eosin-negrosin staining, there was lower sperm viability in varicocele- induced rats, with the highest sperm mortality seen in the 8 month varicocelized animals. The percentage of viable sperm was also higher in the right varicocele group when compared with the left varicocele group. Data for sperm count and morphology are presented in table 2.

Results from the comet assay showed an elevated ratio of sperm DNA fragmentation in the varicoceleinduced animals that increased over time. Sperm from the left varicocelized testes had higher DNA fragmentation when compared with the right testes in the same animals and the control-sham group (Fig 5).

Acridine-orange staining showed that the number of sperm with double-strand DNA breaks was significantly (p<0.05) higher in the varicocelized groups with the highest rate of DNA breaks seen in rats with 8 months varicocelized testes. The percentage of DNA breaks was significantly (p<0.05) lower in sperm from the right testes of varicocelized animals when compared to samples from the left testes (Fig 6). The numerical data for sperm DNA fragmentation and breaks are presented in table 3.

The results for in vitro fertilization(IVF) of oocytes by sperm collected from the right and left testes of varicocelized animals were remarkably lower than the control-sham group (Fig 7A, B). The considerable point was that most of the 2-cell embryos arrested at the first cleavage stage and did not continue further in the varicocelized group. This phenomenon was at its highest rate in samples from the left testes of the varicocelized animals for 8 months. The results for sperm collected from the upper side of the tube (superficial sperm) in terms of progress in cell division were significantly (p<0.05) higher than those collected from the lower side of the tube (sediment sperm). A comparison of the values for fertilization rates between all test groups indicated that the highest value belonged to oocytes exposed to sperm from the right testes of varicocelized animals for 4 months. Therefore, in varicocelized animals the rate of the morula and blastocysts phases compared to the control-sham group declined significantly (p<0.05). The data for IVF rates are presented in table 4.

Results indicated that both TAOC and TTM significantly (p<0.05) decreased after varicocele induction for 4, 6 and 8 months compared to the control-sham group. Accordingly, animals in the 8 month varicocele-induced group showed remarkable diminished antioxidant capacity of serum (Fig 8 A, B).