Ten mature male mongrel dogs that were six to seven months of age and weighed 25.62±2.01 kg (mean±standard deviation) were used in the present study. All dogs were determined to be in good physical condition based upon a physical examination. Experimental dogs were kept in individual pens and received standard balanced canine diets throughout the experiment. All animals were housed for at least seven days before the day of the experiment to adapt to the environment. Animals were kept on a 12-hour light/dark cycle under controlled environmental conditions. Prior to the day of the surgery, food was withheld for at least 12 h before surgery, but the dogs were allowed free access to water. Experimental procedures were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at the Konkuk University (approved No. KU15187).

Experimental dogs received acepromazine (0.1 mg/kg; IM) for premedication and propofol (6 mg/kg; IV) for induction of anesthesia. Endotracheal intubation was performed, and anesthesia was maintained via the inhalation of a combination of isoflurane and oxygen. Physiologic monitoring of animals during surgery and anesthesia included continuous pulse oximetry, ECG, heart and respiratory rates, and body temperature via an esophageal probe. The entire abdomen, from xiphoid to scrotum, was prepared under aseptic conditions. The urinary bladder was emptied via catheterization. Dogs were positioned in dorsal recumbency on a tilting table with the head tilted downward. To surgically induce unilateral or bilateral cryptorchidism, a 4 cm long paramedical incision was made through the skin in the inguinal region. One or two testes were pushed out through the incision, and the fascial attachment between the tail of the epididymis and the scrotum was cut (Figure 1A). The superficial and internal inguinal ring was exposed and enlarged by incising it, and the testicle was introduced into the same side abdominal cavity (Figure 1B). The inguinal ring was closed to prevent the normal descent of the testicle (Figure 1C). After suturing and disinfecting the incision sites, all animals received intramuscular cefazolin sodium (22 mg/kg) for 5 days. In the unilateral group, shamoperated scrotal testes were used as a control. Abdominal and scrotal testes were removed and decapsulated for histological and immunohistochemical analysis 7, 14, 21, and 30 days after surgery. In the orchiopexy group, two dogs underwent bilateral cryptorchidism. After 30 days, the abdominal testes were returned to the scrotum through an inguinal incision and sutured to connect the testes to the scrotum (Figure 1D). Four and eight weeks later, testes were removed and assessed as described above.

Testes and epididymis samples from unilateral cryptorchidism and orchiopexy groups were fixed in Bouin's solution (HT10132; Sigma-Aldrich, St. Louis, MO, USA) overnight at 4℃. Samples were subsequently washed in 70% to 100% (v/v) ethanol, embedded in paraffin, sliced into 5 µm thick sections using a microtome (Thermo, Barrington, IL, USA), and mounted onto glass slides. The mounted canine testis tissue was rehydrated using xylene and 100 to 50% ethanol. The nuclei and cytoplasm were stained with hematoxylin and eosin (H & E). To determine the location of PGP9.5, GATA4, and SCP3 in the testis cells, antigens were retrieved by boiling for 30 min in a tris-ethylenediaminetetraacetic acid (EDTA) solution (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH 9). The antigen-retrieved tissues were membrane permeabilized with PBS containing 0.1% Triton X-100 for 10 minutes. Non-specific protein binding was blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour at room temperature (RT), and tissues were incubated overnight at 4℃ with the following primary antibodies: anti-PGP9.5 antibody (1:1000; 7863- 1004; AbD Serotec, Raleigh, NC, USA), anti-GATA4 antibody (1:50; SC-1237; Santa Cruz Biotechnology, Inc.; Dallas, Texas, USA), and anti-SCP3 antibody (1:50; SC-27333; Santa Cruz Biotechnology, Inc.; Dallas, Texas, USA). After washing three times with PBS, the appropriate secondary antibodies were added for two hours at room temperature. The peroxidase substrate detection kit (SK-4100; Vector Laboratories) was used for detection of the putative canine germ cell markers according to the manufacturer's instructions. For each testis, the diameter of seminiferous tubules in ten randomly selected tubular profiles that were round or nearly round, were measured under light microscopy.

The diameter of seminiferous tubules and number of PGP9.5, and GATA4 positive cells were analyzed by ANOVA and Tukey,s tests using Graphpad Prism 5 software (Graphpad, LaJolla, CA, USA). with P<0.05 considered statistically significant. The mean and standard deviation (SD) were calculated for each value.

To evaluate the effect of surgically induced unilateral cryptorchidism, H & E staining was performed. The histological appearance and diameter of the seminiferous tubules in the scrotal and cryptorchid testes of dogs 7, 12, 21, and 30 days following surgical induction of cryptorchidism was analyzed (Figure 2). No significant change in seminiferous tubule diameter was observed in the scrotal testes measured at different days (Figure 2). Surgical induction of cryptorchidism significantly decreased the diameters of the seminiferous tubules compared to those of the scrotal testis by 82% in 7 days, 69% in 14 days, 71% in 21 days, and 45% in 30 days (P<0.01) (Figure 2B). After thirty days from induction of cryptorchidism, the operated testis was markedly smaller than the scrotal testis (Figure 3A), and sperm were not found in the cauda epididymis of cryptorchid testis (Figure 3B) compared with scrotal testis (Figure 3C).

To identify the changes of cell number in the canine testes after surgically induced cryptorchidism, immunohistochemistry was used. Expression of PGP9.5 was detected to identify undifferentiated spermatogonia, and expression of GATA-binding protein 4 was detected to recognize Sertoli cells in the scrotal and cryptorchid testes. PGP9.5-positive spermatogonia at days 7, 14, 21, and 30 after surgery are shown in Figure 4A. Abdominal testes exhibited significantly lower numbers of spermatogonia than did scrotal testes, (Figure 4B). On day 30 after inducing cryptorchidism, the numbers of spermatogonia in the abdominal testes reached the lowest level, averaging 2.6±0.89. This was significantly lower than the average numbers of 29.4±1.4 as measured in the control testes (Figure 4B). Sertoli cells were visualized using the GATA4 marker, and no significant difference was observed in the numbers of GATA4 positive cells in scrotal and cryptorchid testes 7,14, 21, and 30 days after surgically induced cryptorchidism (Figure 5).

To confirm the regeneration of germ cells in cryptorchid testes after orchiopexy procedures, H & E staining and immunohistochemistry analysis were performed. Differentiated germ cells were detected in orchiopexy testes 4 weeks after orchiopexy (Figure 6A, D). However, sperm was not detected in orchiopexy testes and epididymis 4 weeks after orchiopexy (Figure 6A, B). Immunohistochemistry results showed PGP9.5 positive un-differentiated spermatogonia and SCP3 positive primary spermatocytes in seminiferous tubules of orchiopexy testes (Figure 6C, D). After eight weeks from orchiopexy, attempts at manual semen collection succeeded in all two dogs (Figure 6E).