Expression of Protein Markers in Spermatogenic and Supporting Sertoli Cells Affected by High Abdominal Temperature in Cryptorchidism Model Mice

Abstract

Cryptorchidism is a congenital abnormality resulting in increased rates of infertility and testicular cancer. We used cryptorchidism model mice that presented with the translocation of the left testis from the scrotum to the abdominal cavity. Mice underwent the surgical procedure of the left testis at day 0 and were sacrificed at days 3, 5, 7, 14, 21, and 28 post-operatively. The weight of the left cryptorchid testis decreased significantly at days 21 and 28. The morphological changes were observed after 5 days and showed detached spermatogenic cells and abnormal formation of acrosome at day 5, multinucleated giant cells at day 7, and atrophy of seminiferous tubules at days 21 and 28. The high abdominal temperature disrupted the normal expression of cell adhesion molecule-1, Nectin-2, and Nectin-3 which are essential for spermatogenesis. In addition, the pattern and alignment of acetylated tubulin in cryptorchid testes were also changed at days 5, 7, 14, 21, and 28. Ultrastructure of cryptorchid testes revealed giant cells that had been formed by spermatogonia, spermatocytes, and round and elongating spermatids. The study’s findings reveal that cryptorchidism’s duration is linked to abnormal changes in the testis, impacting protein marker expression in spermatogenic and Sertoli cells. These changes stem from the induction of high abdominal temperature.

Introduction

Cryptorchidism is a congenital condition in which one or both testes have not descended into the scrotal sac. This condition is one of the common causes of developmental abnormality, linked to male infertility and testicular cancer. ¹ Unilateral cryptorchidism is a condition in which one of the testes remains within the abdominal cavity and the other has passed down into the scrotal sac. The scrotal sac helps to protect and maintains the temperature necessary for spermatogenesis, normally 2–4C lower than the normal body temperature. Although, one testis in scrotal sac can continue normal function in spermatogenesis and steroidogenesis, the total functional rate is reduced. ² The higher temperature in the abdominal cavity affects the normal function of sperm production before complete maturation, and the germ cells form multinucleated giant cells in the seminiferous tubule of the cryptorchid testis. ³

As is widely known, spermatogenesis is the necessary process in the male, essential for sperm production. Spermatogenesis starts with mitotic division of the spermatogonia, located on the basement membrane of the seminiferous tubules. Undifferentiated A spermatogonia divide to make more stem cells, whereas differentiated A spermatogonia proliferate and develop respectively to intermediate and B spermatogonia types.^4,5 Spermatogonia then pass through the mitotic phase to become spermatocytes. Preleptotene cells are the resting spermatocytes, which go through the S-phase of the cell cycle, to become leptotene, zygotene, pachytene, and diplotene primary spermatocytes, in the prophase of meiosis I (M-I). Diplotene is the last phase of the primary spermatocyte, which then proceed through prophase, metaphase, anaphase, telophase, and then finally complete the cell division of M-I. Secondary spermatocytes divide rapidly to follow the process of meiosis II and produce spermatids. ⁴ There are two types of spermatids, namely round and elongating spermatids. Spermatids start to form acrosomes and develop flagellum during spermiogenesis. The final step is when elongating spermatids transform into spermatozoa within the luminal compartment of the seminiferous tubules. This progression of the spermatids can be classified using immunofluorescence (IF) staining of peanut agglutinin (PNA) which is represented specifically with acrosomal formation during spermiogenesis.^6,7

Cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily, known as the spermatogenic immunoglobulin superfamily, that consists of an extracellular domain containing three immunoglobulin-like loops, transmembrane, as well as short intracellular domains. Cadm1 is localized on the plasma membrane of spermatogenic cells, which binds heterophilically to poliovirus receptor (PVR) on a Sertoli cell and also bind homophilically to Cadm1 on other spermatogenic cells. ⁸ Cadm1 expressed in the plasma membrane of intermediate spermatogonia to early pachytene spermatocytes, Steps 7–16 spermatids, and the caudal portion of elongating spermatids. Cadm1 is not expressed in round spermatids, mature spermatozoa, and Sertoli cells.^7,8 Cadm1 has a crucial role in spermatogenesis. Cadm1-deficient mice have obviously reduced numbers of elongating spermatids, reveal abnormal morphology of elongating spermatids, and reveal detached germ cells present in the epididymis. ⁹ However, the functional relation between spermatogenic cells and Sertoli cells is important in the development of germ cells to form spermatozoa in the seminiferous tubules. Other cell adhesion molecules that are involved with this interaction of spermatogenic cells and Sertoli cells are Nectin-2 and Nectin-3. These are the Ca²⁺ concentration (Ca²⁺)-independent immunoglobulin-like cell–cell adhesion molecules that are expressed in the plasma membrane of Sertoli cells and spermatids, to form the Sertoli–spermatid junction for spermatid development.^10,11 Nectin-2 is located in the Sertoli cell membrane, whereas Nectin-3 is expressed in the spermatid membrane. Nectin-2 is localized both in the Sertoli–Sertoli junctions of the basal regions (basal ectoplasmic specialization (ES)) and also interacts with Nectin-3 at the Sertoli–spermatid junctions of the luminal regions (apical ES). ⁹ A deficiency of Nectin-2 results in abnormal shape and vacuolization of nuclei in Step 11–16 spermatids, whereas Nectin-3 knockout mice exhibit expression of Nectin-2 at the basal region of the seminiferous tubules.^9,12,13 Moreover, Nectin-3 disappears in the testes of Nectin-3^−/− mice, presents irregular shapes of spermatids nuclei, and shows abnormal formation of the head and midpiece in spermatozoa.^10,13 The sperm flagellum plays a critical role in sperm movement. When acrosome formation starts, the microtubule-containing manchette assembles alongside the elongating spermatid head. ¹⁴ The manchette forms on the opposite side of the acrosomal surface and induces the development of sperm flagellum.^4,15 Acetylated tubulin (Ac-tubulin) is one of tubulin subtypes that occurs during acetylation on the amino group of lysine-40 at the N-terminal domain of α-tubulin. ¹⁶ In addition, Ac-tubulin has colocalization with the hook microtubule tethering protein 2 (HOOK2) on the microtubules of the manchette and can be detected in the axoneme of spermatozoa. ¹⁷ The protein markers in spermatogenic cells do not only play an important role, supporting Sertoli cells also help in the process of spermatogenesis. Wilms’ Tumor 1 (WT1) is expressed in the nucleus of the Sertoli cell and is also required for testosterone production in the Leydig cells. ¹⁸ WT1 expresses in the nucleus and activates the actin remodeling at the cell membrane, essential for migration of germ cells and maturation. Moreover, WT1 increases the level of importin-alpha1 that informs the nucleus to import proteins for spermatogenesis. ¹⁹ Previous studies reported that WT1 encodes the important transcription factor for genital tract development. WT1 functions in the survival and proliferation of cells in the genital ridge and induces somatic cells become Sertoli cells.^20,21

The higher intra-abdominal temperature is one of the factors that disrupt the normal processes of spermatogenesis and contributes to infertility in males. However, this disruption mechanism is still unclear and cannot explain the order in the abnormal phase. We demonstrated the morphological appearance of cryptorchid testes and epididymis, presented the expression of protein markers in spermatogenic and supporting Sertoli cells, and observed the ultrastructure of cells by transmission electron microscope (TEM). Hence, the aim of this study was to explain the altered expression of protein markers in abnormal spermatogenic and supporting Sertoli cells caused by the higher abdominal temperature.

Materials and Methods

Animals

All experiments were performed on adult male Institute of Cancer Research (ICR) mice (SIc:IRC), 9–13 weeks old, from Japan SLC, Inc., Shizuoka, Japan. The mice were housed in cages with soft bedding and free access to food and water, under a 12-hr light/12-hr dark cycle. The mice were habituated for 3 days before pretesting. All procedures complied with the ethical guidelines of the Animal Care and Use Committee that approves and manages animal experiments performed in Kumamoto University.

A total of 42 male ICR mice (SIc: IRC) were randomized into seven groups (control, 3D, 5D, 7D, 14D, 21D, and 28D). Control group mice underwent only abdominal incision and were sacrificed at day 0. Other group mice (3D, 5D, 7D, 14D, 21D, and 28D groups) underwent a surgical procedure for unilateral cryptorchidism of left testis at day 0 and were sacrificed at days 3, 5, 7, 14, 21, and 28 post-operatively.

Surgical Procedure

Surgical procedure for unilateral cryptorchidism was performed at day 0. Mice were anesthetized with a combination (M/M/B: 03/4/5) of medetomidine (0.3 mg/kg), midazolam (4.0 mg/kg), and butorphanol (5.0 mg/kg) by intraperitoneal injection. ²² A left-sided abdominal incision was made, the left testis was pushed into the peritoneal cavity, and then the gubernaculum was clipped and fixed with the fat pad of the peritoneum to simulate a cryptorchid testis. The right testis was left as the control testis. ²³ After operation, skin was closed by wound clips and medetomidine antagonist (0.75 mg/kg) was administered to speed recovery from general anesthesia. At the experimental endpoint at days 3, 5, 7, 14, 21, and 28 post-operatively, mice were anesthetized with sodium pentobarbital (100 mg/kg body weight) by intraperitoneal injection. Mice were sacrificed by transcardial perfusion with 25 ml of normal saline for each individual.

Histological Analyses

The morphological appearance of the testes and epididymis was studied using hematoxylin and eosin (H&E) staining. Three mice from each group were sacrificed by transcardial perfusion with 25 ml of normal saline and then injected with 15 ml of 4% paraformaldehyde into the apex of heart at the experimental endpoint. The testes and epididymis were collected and fixed in 4% paraformaldehyde at 4C and left overnight. Specimens were prepared in paraffin before transverse cutting at a thickness of 4 micrometers by the microtome and the sections were mounted on adhesive glass slides. Cross sections of the testis and the epididymis were viewed under an Olympus BX51 microscope (Nagano Olympus Co., Ltd., Tokyo, Japan).

IF Staining

The sections were deparaffinized and dehydrated, and routinely incubated in antigen retrieval (Tris-HCl buffer 20 mM, pH 9.0) at 95C for 15 min before proceeding with the IF staining protocol. The heat-induced antigen retrieval helps to enhance the ability of a primary antibody to bind to a specific protein in formalin-fixed paraffin-embedded tissues. ²⁴ After incubation, the sections were kept in the buffer and allowed to cool down naturally to room temperature for 3 hr. The sections were then washed twice in phosphate-buffered saline (PBS) for 3 min each and the nonspecific proteins blocked by incubating the sections with 1% bovine serum albumin in PBS for 30 min at room temperature. They were then incubated overnight at 4C with the following primary antibodies (Appendix Table 1). The reactions were visualized by incubation for 1 hr at room temperature with the following secondary antibodies (Appendix Table 1). For visualization of acrosomes, sections were incubated with PNA conjugated to Alexa Fluor 488 (green; 1:500; Molecular Probes, Eugene, Oregon, USA) for 30 min, and for stainability of DNA in nuclei were incubated with bisbenzimide H33258 (blue; Hoechst 33258, Sigma-Aldrich, St Louis, Missouri, USA) in a dilution of 1:2000 for 10 min at room temperature. The sections were viewed under a Keyence BZ-X700 all-in-one fluorescence microscope (Keyence corporation of America, Itasca, Illinois, USA).

Percentage of Apoptosis per One Seminiferous Tubule

Caspase-3 is a marker for programmed cell death (apoptosis). The sections were incubated with rabbit monoclonal anti-cleaved Caspase-3 (Asp175; 5A1E) primary antibody (1:400; 9664, Cell signaling technology, Danvers, Massachusetts, USA) overnight at 4C and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red; 1:400; ab150080, Abcam, Cambridge, UK) for 1 hr at room temperature. For visualization of cleaved Caspase-3 (Cle-caspase-3)-positive, the cells were viewed under a Keyence BZ-X700 all-in-one fluorescence microscope and a photo was taken at magnification of 40×. The percentage of apoptosis was presented within the area of seminiferous tubules proportional to Caspase-3 expression. The number of seminiferous tubules from 90 figures was averaged for each sample (right and left testes) in each animal.

Transmission Electron Microscopy

The testes were collected, tissues cut to a 2-mm³ size and fixed immediately in ½ Karnovsky, 2% paraformaldehyde/2.5% glutaraldehyde/0.1 M phosphate-buffered PB (pH 7.4) for 2 hr at 4C. Then specimens were washed and post-fixed in 1% osmium tetroxide for 1 hr at 4C. Specimens were prepared in epoxy resin before transverse cutting at a thickness of 1 micrometer by the ultramicrotome (Leica EM UC7, Leica Microsystems CMS GmbH, Wetzlar, Germany). The semithin sections were stained with 1% Toluidine blue stain. The ultrathin sections (0.1-micrometer thick) were cut by diamond knife and stained with 1% uranyl acetate. The stained ultrathin sections of the testes were viewed under a TEM HT7700 (Hitachi High-Tech Corporation, Tokyo, Japan).

Statistical Analyses

All numerical data were presented as mean ± standard error of the mean. Statistical analysis of any significant differences between multiple group means was tested using One-way ANOVA, followed by Tukey’s Honest Significant Difference post hoc test when differences were observed. Paired sample t-test was used to compare means between right (normal) and left (cryptorchid) sides in each group. In all statistical comparisons, differences were considered significant at p-value <0.05.

Results

Morphological Appearance of Normal and Cryptorchid Testes

The testis consists of several long tubules which are the site for spermatogenesis, known as seminiferous tubules. A cross section of a testis reveals the interstitial tissue between the seminiferous tubules. The main component of interstitial tissue is collagen fibers and Leydig cells, including some fibroblasts and a few blood vessels. Each seminiferous tubule consists of spermatogenic cells and supporting Sertoli cells. Spermatogonia and preleptotene spermatocytes are attached to the basal laminar of the tubules. During the process of spermatogenesis, the Sertoli cells help the developing spermatogenic cells to move away from the basal laminar to the lumen of the tubule, passing through the tight junctional complexes between the Sertoli cells and spermatogenic cells. Sertoli cells lie on the basal laminar and extend their cytoplasm to the luminal surface of the seminiferous tubule. The nucleus of a Sertoli cell is ovoid and lightly stained in color compared with the nucleus of spermatogenic cells.

The morphological abnormalities in cryptorchid testis started to be revealed at 5D. The seminiferous tubules of the 5D group showed detached germ cells in the lumen of the seminiferous tubules (Fig. 1E and F). Cryptorchid testis in 7D group revealed multinucleated giant cells in the lumen and represented the failure of sperm release (Fig. 1G and H). The Sertoli cells may have lost their function of moving elongating spermatids to the apical compartment and then release into the tubular lumen. At 14D, a multinucleated giant cell was formed from abnormal spermatids. There are the large spaces that resulted in the loss of cell–cell interaction (Fig. 1I and J).

Figure 1.

Light microscopic images (H&E staining) of the normal and cryptorchid mouse testes. Control (Panels A and B) and 3D (Panels C and D) groups had an organized arrangement of spermatogenic cells and supporting Sertoli cells. 5D group shows spermatids and detached germ cells in the lumen of the seminiferous tubule (Panels E and F). The multinucleated giant cells (arrow) of the tubule and the failure of sperm (arrowhead) release appeared at 7D (Panels G and H). Left testis of 14D group reveals a multinucleated giant cell (arrow) that was created by a formation of abnormal spermatids (Panels I and J). The large space that appeared around the cells (arrowhead) resulted in the loss of cell–cell interaction (Panel J). Elongating spermatids were lost, multinucleated giant cells (arrow) were detected, and the large vacuole (V) due to the shedding of cells from the seminiferous epithelium appeared in 21D and 28D groups (Panels K to N). Abbreviation: H&E, hematoxylin and eosin. (Bars A, C, E, G, I, K, M = 50 micrometers; bars B, D, F, H, J, L, N = 20 micrometers).

The diameter of the seminiferous tubules in the 21D and 28D groups was smaller than the control for the reason that the light microscope images revealed many atrophic tubules in cryptorchid testes in both 21D and 28D groups. The elongating spermatids disappeared at 21D and afterward. Multinucleated giant cells are also detected. The large vacuole due to the shedding of cells from the seminiferous epithelium appeared at 21D and 28D (Fig. 1K to N). Also seen are degenerating spermatocytes, degenerating spermatids, and apoptotic cells. The abnormal formation of acrosomal pattern and the delayed development of acrosomes resulted the formation of many abnormal spermatids.

The overview ultrastructure of cells in the seminiferous tubule did not show in the picture result. The sample of the 5D cryptorchid testis represented that some spermatids had a small vacuole close to their nuclei at the apical compartment of the tubule. Abnormal formation of acrosomes in Step 6 was observed by TEM. There was a multinucleated giant cell resulting from three B spermatogonia lying on the basal lamina. Some Sertoli cells showed vacuoles in their cytoplasm and mitochondria. The vacuolization in the cytoplasm was caused by endoplasmic reticulum swelling. In addition, a degenerating spermatid was identified at 5D. Furthermore, the multinucleated giant cell resulting from two abnormal spermatocytes was observed by TEM and seen for the first time at 7D. The head and flagellum of an elongating spermatid were detected in the cytoplasm of a Sertoli cell. Some elongating spermatids, which showed abnormal formation of both the sperm head and developing flagellum, were phagocytosed by the large vesicle.

At 14D, the ultrastructure of the cryptorchid testis showed two spermatids fused into the giant cell, as well as a few apoptotic cells. The degenerating spermatocytes and apoptotic cells were also detected in the seminiferous tubule. In addition, there were a lot of lipid droplets and residual bodies in the seminiferous tubules. Moreover, abnormal head formation of an elongating spermatid presented with vacuolization and an abnormal shape of the acrosome, as well as an irregular shape of the sperm head.

The samples of 21D and 28D cryptorchid testes revealed that the cytoplasm of the Sertoli cells showed vacuolization, as well as a lot of multivesicular bodies, referred to as abnormal mitochondria. We also observed the ultrastructure of the basal lamina and peritubular myoid cells. The irregular collagen fibers in the basal lamina and lysosome-bound nanoparticles in the peritubular myoid cells were identified in the cryptorchid testis. At 28D, degeneration of giant cells occurred via a process of microautophagy and presented as myelin-like structures in the cell. Some B spermatogonia were detached from the basal lamina but still connected to adjacent cells by the intercellular bridge. Mitochondrial swelling was also detected in the cytoplasm of Sertoli cells and revealed many degenerating cells, vacuolization, microautophagy, including apoptotic cells. Most vacuolization occurred by 28D post-operatively. However, the overall ultrastructure of the cryptorchid testis at 28D was similar to 21D.

Detached Germ Cells and Giant Cells in Left Epididymis

H&E staining was used for histological study of the epididymis. The epididymis is the site of sperm maturation and is lined by a pseudostratified columnar epithelium with stereocilia. The epithelium is composed of two types of cells, regenerative basal cells and stereociliated principal (columnar) cells. The wall of the epididymis contains circular layers of smooth muscle whose peristaltic contractions facilitate the delivery of sperm into the ductus deferens.

The caput of the epididymis in 3D groups shows a lot of sperm in the lumen, similar to the control group. Sperm levels in the epididymis at 5D and 7D were lower than the control and revealed some detached germ cells and debris in the lumen. Sperm numbers in the left epididymis decreased gradually post-operatively in the cryptorchidism model. On the other hand, the epididymis of 14D, 21D, and 28D groups shows gradually increased numbers of detached germ cells, giant cells, and debris in the lumen. However, the weight of left epididymis was not significantly altered when compared with the left-side control or same-side control epididymis.

Ultrastructure of Giant Cells

A TEM was used to observe the ultrastructure of normal spermatogenic cells in the normal control testis and the multinucleated giant cells in the cryptorchid testis (Fig. 2). In the normal control testis, spermatogonia lay on the basal laminar in the basal compartment of the seminiferous tubule. The type A spermatogonium has an ovoid nucleus with a moderately dense heterochromatin and shows a reticulated nucleolus (Fig. 2A). Spermatogonia renewal and proliferation involve passing through a mitotic phase to become spermatocytes. The pachytene spermatocyte reveals a protein structure that forms between homologous chromosomes, known as the synaptonemal complex, and the sex vesicle containing the sex chromatin, in the first meiotic prophase stage (Fig. 2B). An acrosome derives from the Golgi apparatus and appears close to the nucleus of the round spermatid. The pattern of acrosomal formation and diffusion of degree angle are used to classify the stepwise formation of the spermatid. Step-6 spermatid appears to have an acrosomal cap covering one fourth of the nuclear surface and possesses a dense structure of RNAs and RNA-binding proteins, known as the chromatoid body, in the cytoplasm (Fig. 2C). Elongating spermatid has an acrosome cap that continues to elongate, covering the nucleus at the head portion (Fig. 2D).

Figure 2.

Electron microscopic images of normal spermatogenic cells and multinucleated giant cells. Spermatogenic cells of control testis (Panels A to D). Spermatogenic cells of cryptorchidism (Panels E to J). Spermatogonium lying on basal laminar (Panel A). Pachytene seen in the synaptonemal complex (Syn) and sex vesicle (Sv) in the nucleus (Panel B). Step-6 spermatid shows an acrosome that is derived from the Golgi apparatus (G) and also shows a chromatoid body (Cb) in the cytoplasm (Panel C). Elongating spermatid reveals an acrosome (arrowhead) at the head portion (Panel D). Multinucleated giant cell consisting of many heads and flagellums (arrowhead) of elongating spermatids (Panel E). Abnormal elongating spermatid shows vacuolization in the nucleus and swollen mitochondria (arrowhead) in the residual body (Panel F). Four spermatogonia fused to form multinucleated giant cells (Panel G). The multinucleated giant cell resulted from the fusion of two degenerating spermatocytes that are present in the synaptonemal complex (Syn) of the nucleus and had many swollen mitochondria (arrowhead) in the cytoplasm (Panel H). The multinucleated giant cells formed from two abnormal round spermatids and apoptotic cells, then fused into one large cell (Panels I and J). Abbreviations: A, acrosome; Bl, basal laminar; H, head; M, mitochondria; P, peritubular myoid cell (arrowhead); N, nucleus; Ss, subacrosomal space; V, vacuole. (Scale bars = 500 nm).

The higher abdominal temperature disrupted the normal development of sperm before complete maturation. Abnormal spermatogenic cells lack the interaction with Sertoli cells and become isolated germ cells or fused to form the multinucleated giant cells. The multinucleated giant cell appeared for the first time in cryptorchid testis at 5D. A multinucleated giant cell formed from spermatogonia, spermatocytes, and round and elongating spermatids was detected. Spermatids with absent acrosome also can form a giant cell. There were many heads and flagellums of elongating spermatids that had fused, with some degenerating cells, into one large cell (Fig. 2E). Abnormal elongating spermatids revealed vacuolization of the nucleus. The subacrosomal space can be visualized in the area between the developing acrosome and the elongated nucleus. The residual body of the abnormal elongating spermatid contains a large vacuole and a lot of swollen mitochondria (Fig. 2F). The multinucleated giant cell in the basal compartment resulted from the spermatogonia lacking the intercellular bridge (Fig. 2G). The ultrastructure of cryptorchid testes in 14D group showed a multinucleated giant cell, resulting from the fusion of two degenerating spermatocytes, present in the synaptonemal complex of the nucleus and had many swollen mitochondria in the cytoplasm (Fig. 2H). The delayed development of the acrosome in some spermatids can be observed in the cryptorchid testis at 5D. These abnormal round spermatids tended to move and fuse, to form a multinucleated giant cell following the long-time period of cryptorchidism (Fig. 2I). Auto-phagocytosis occurred in the multinucleated giant cell and was detected at 28D. Moreover, swollen mitochondria and apoptotic cells were also detected in the cytoplasm of this giant cell (Fig. 2J).

Localization and Expression of Cadm1

A previous study reported that Cadm1 is the cell adhesion molecule and is expressed in the plasma membrane of intermediate spermatogonia to early pachytene, spermatocytes Steps 7–16 spermatids, and in the caudal portion of elongating spermatids.^7,8 The observation of Cadm1 expression in this study found that it is located in the plasma membrane of germ cells, located in the basal compartment of the tubule, as well as in the apical compartment of the caudal portion of elongating spermatids (Fig. 4). We used the PNA lectin to identify the acrosome and used to divide the stages of the seminiferous tubule cycle. IF of Cadm1 reactivity in Stage IV of seminiferous tubule decreased in the caudal portion of elongating spermatids in cryptorchid testes at 5D, 7D, and 14D, whereas it was constant in spermatogonia and spermatocytes. In addition, PNA expression in 5D and 7D groups also showed the delayed development of the acrosome. The higher abdominal temperature affected the cryptorchid testes in both 21D and 28D groups, loosing elongating spermatids and cell–cell connection. Cadm1 expression in spermatogonia and spermatocytes also decreased at 21D and 28D. Moreover, Cadm1 was not expressed in detached germ cells and giant cells (Fig. 3). We observed expression of Cadm1 in the multinucleated giant cell of cryptorchid testis at 7D and 21D. Cadm1 expression was still present in germ cells at 7D, minimally present at 21D but not detected in giant cells, both at 7D and at 21D groups (Fig. 5).

Figure 4.

Fluorescence images of Stage XI of the mouse seminiferous tubules showed the expression of Nectin-2 (green) and Nectin-3 (red) in the control group and cryptorchidism groups (3D, 5D, 7D,14D, 21D, and 28D). Nuclei were counterstained with Hoechst (blue). Nectin-2 and Nectin-3 revealed coexpression as a yellow color (arrow) at the binding sites of Sertoli–Spermatid cells at the apical compartment of the tubule. Nectin-2 was expressed both in the apical and in the basal (arrowhead) compartments of tubules in control. Nectin-2 expression started to decrease in the apical compartment at 5D. Nectin-3 expression in the apical compartment was maintained until 7D but decreased after 14D. Nectin-3 also expressed slightly in some round spermatids in 21D and 28D groups. (Scale bars = 50 micrometers.)

Figure 3.

Fluorescence images of Stage IV of mouse seminiferous tubules showed the expression of Cadm1 (red) in control group and cryptorchidism groups (3D, 5D, 7D, 14D, 21D, and 28D). Peanut agglutinin (PNA; green) lectin was used to identify acrosome at head of spermatids and nuclei were counterstained with Hoechst (blue). Cadm1 expressed in basal compartment of seminiferous tubule and caudal portion of elongating spermatids (arrow head) at 3D same as control group. Cadm1 expression decreased in the caudal portion of the elongating spermatids in cryptorchid testes at 5D, 7D, and 14D, whereas it was constant in spermatogonia and spermatocytes and did not express in detached germ cell (14D; arrow). At 21D and 28D, Cadm1 also decreased in both spermatogonia and spermatocytes, and did not express in giant cells (arrow). (Scale bars = 50 micrometers.)

Figure 5.

Expression of adhesion molecules (Cadm1 and Nectin-3) in the multinucleated giant cells of cryptorchid testis at 7D and 21D. The multinucleated giant cells appeared in both 7D and 21D groups stained by H&E. The expression of Cadm1 is present in germ cells but not detected in giant cells at 7D and 21D. Nectin-3 was seen in the head portion of the elongating spermatids that fused to form the multinucleated giant cell in 7D group. 21D group had absent elongating spermatids and zero Nectin-3 in the multinucleated giant cell. Abbreviation: H&E, hematoxylin and eosin. (Bars H&E = 20 micrometers; fluorescence = 50 micrometers.)

Localization and Expression of Nectin-2 and Nectin-3

Nectin-2 and Nectin-3 are the Ca²⁺-independent immunoglobulin-like cell–cell adhesion molecules that are expressed in the plasma membrane of cells. When using the 40× magnification for observation Nectin-2 was found at the basal and apical compartments in both control and 3D groups (Fig. 4). In the basal compartment, Nectin-2 was present at the junction of the Sertoli–basal laminar and the junction between Sertoli and Sertoli cells at the blood testis barrier, referred to the basal ES. Not only was Nectin-2 expressed at the apical compartment, but also Nectin-3. Nectin-3 was expressed in the plasma membrane of the head portion of spermatids. Double IF staining between Nectin-2 and Nectin-3 revealed the coexpression (yellow color) of Nectin-2 or Nectin-3 at the binding sites between the apical part of Sertoli cells and the head of spermatids. The expression of Nectin-2 in the cryptorchid testis started to decrease at the binding sites with Nectin-3 at 5D. Similarly, the expression of Nectin-3 decreased after 5D. In the cryptorchid testes of 5D and 7D groups, the interaction between Nectin-2 and Nectin-3 was seen in elongated spermatids that showed normal head position in the tubule. If the head position of elongated spermatids faced up to the lumen, they expressed only Nectin-3. At 21D and 28D, Nectin-2 interacted with Nectin-3 in the plasma membrane of some round spermatids, some germ cells, and some giant cells. In addition, Nectin-3 expression translocated from plasma membrane into the nucleus in some round spermatids. Nectin-2 decreased greatly in the apical compartment at 28D. However, Nectin-2 still had strong expression at the basal compartment in cryptorchid testes, whereas Nectin-3 decreased severely at 21D and 28D (Fig. 4). We observed the expression of Nectin-3 in the multinucleated giant cells of the cryptorchid testis at 7D and 21D. Nectin-3 was seen in the head portion of the abnormal elongating spermatids, fused to form the multinucleated giant cell in 7D groups. In contrast, 21D group had absent elongating spermatids and zero Nectin-3 in the multinucleated giant cell (Fig. 5).

Localization and Expression of Ac-Tubulin

Ac-tubulin is an α-tubulin acetylation on the amino group of lysine-40 at the N-terminal domain. Ac-tubulin is localized both in the manchette and in the axoneme of developing sperm. We studied the localization of Ac-tubulin in normal testis, using double IF with vimentin staining (Fig. 6). Vimentin detected in the cytoplasm of Sertoli cells and was also strongly expressed surrounding the nucleus. Ac-tubulin was expressed in the axoneme of elongating spermatid, also the apical compartment of Sertoli cells and was also expressed in the seminiferous epithelium. The pattern and alignment of Ac-tubulin in the cryptorchid testes changed at 5D, 7D, 14D, 21D, and 28D. At 5D and 7D, Ac-tubulin was expressed in some manchette and shown as a short tubular pattern at the original site of the axoneme. At 14D, Ac-tubulin disappeared in the axoneme and was shown as a circular pattern in some spermatids. Moreover, Ac-tubulin was strongly expressed in meiotic cell division and was detected in giant cells at Stage XII of the seminiferous tubule of 21D group. However, Ac-tubulin was identified in the cytoplasm of Sertoli cells and strongly expressed in the seminiferous epithelium, particularly in spermatids, at 28D (Fig. 6).

Figure 6.

Fluorescence images of mouse seminiferous tubules show the expression of acetylated tubulin (Ac-tubulin; red). Vimentin (green) was used to identify Sertoli cells in the control. Peanut agglutinin (PNA; green) lectin was used to identify the acrosome at the head of spermatids and nuclei were stained with Hoechst (blue). Vimentin stains strongly in the cytoplasm surrounding the nucleus of Sertoli cells. In the control, Ac-tubulin is present in the apical compartment of the Sertoli cells, axoneme of elongating spermatid, and the opposite side of acrosomal surface in Step 9 or 10 spermatids, the same as 3D. The pattern and alignment of Ac-tubulin changed at 5D, 7D, 14D, 21D, and 28D. At 14D, Ac-tubulin disappeared in the axoneme and presented as a circular pattern (arrow) in some spermatids. At Stage XII of seminiferous tubule in 21D group, Ac-tubulin expressed strongly in meiotic division cell (arrow) and was detected in giant cell (arrowhead). Ac-tubulin was identified in the cytoplasm of Sertoli cells (arrowhead) and in the remaining round spermatids at 28D. (Bars A, C, E, G, I, K, M = 20 micrometers; bars B, D, F, H, J, L, N = 50 micrometers).

Localization and Expression of WT1

WT1 is a tumor-suppressor gene function in transcription factor and essential for normal development of the urogenital system. WT1 was detected in the nucleus of mature Sertoli cells in the adult mouse testis. The WT1 IF staining in the cryptorchid testes, after the operation, was to determine the effects of higher abdominal temperature on Sertoli cells. Stage IV of the seminiferous tubule showed that WT1 was expressed in the nucleus of mature Sertoli cells. The number of WT1-positive Sertoli cells per tubule in 14D, 21D, and 28D groups was high, whereas it was close to normal in 3D, 5D, and 7D groups when compared to control (Fig. 7).

Figure 7.

Fluorescence images of mouse seminiferous tubules showed the expression of WT1 (red) in control group (0D: Panels A and H) and cryptorchidism groups (3D: Panel B, 5D: Panel C, 7D: Panel D,14D: Panel E, 21D: Panels F and I, and 28D: Panels G and J). Peanut agglutinin (PNA; green) lectin was used to identify acrosome at the head of spermatids and nuclei were counterstained with Hoechst (blue). The cell proliferation (arrowhead) occurred in the basal compartment and was detected by BrdU (green; Panels H to J). Stage IV of seminiferous tubule showed that WT1 expressed in mature Sertoli cells (Panels A to G). Stage II or III of seminiferous tubule showed that BrDU was able to be detected in the nucleus of undifferentiated A spermatogonia and intermediate spermatogonia (Panels H to J). The double immunofluorescence in the cryptorchid testis at 21D and 28D did not show coexpression between WT1 and BrdU (Panels I and J). The graph showed that the diameter of seminiferous tubule decreased significantly, whereas the number of WT1-positive cells increased significantly at 14D, 21D, and 28D, compared with control. *p<0.05. (Scale bars = 50 micrometers.)

BrdU is the common marker for identifying cell proliferation and does not express in mature Sertoli cells. BrdU was detected in the nucleus of undifferentiated A spermatogonia and intermediate spermatogonia. In Stage II or III, double IF of WT1 and BrdU in cryptorchid testis had no coexpression at 21D and 28D. So the mature Sertoli cells, in both control and cryptorchid testes, were non-proliferative cells. Moreover, the graph of diameter of seminiferous tubule showed that the column of cryptorchid testes decreased significantly at 14D (202.600 ± 5.489), 21D (187.252 ± 4.594), and 28D (207.858 ± 6.027), compared with control (224.549 ± 3.906). On the other hand, the number of WT1-positive cells per tubule increased significantly at 14D (19.767 ± 0.893), 21D (20.167 ± 0.656), and 28D (20.267 ± 0.719) when compared with control (14.867 ± 0.377). Therefore, the number of WT1-positive Sertoli cells had a negative correlation with the diameter of seminiferous tubule using the Pearson correlation coefficient.

Cle-Caspase-3 and Apoptosis

Programmed cell death or apoptosis was detected by caspase-3. Caspases are synthesized as inactive precursors (procaspases). Procaspase can become active and cleaves by several mechanisms including effector caspase-3. The activated caspase-3 used in this study was a result of cleavage adjacent to Asp175. In control testis, Cle-caspase-3 expressed slightly in the basal compartment of the tubule, the same as 3D group. Strong expression of Cle-caspase-3 in some germ cells started to be revealed at 5D. At 7D, Cle-caspase-3 was able to be detected in germ cells, spermatocytes, and spermatids. Moreover, cells in the interstitial tissue, which were referred to as Leydig cells, also showed expression of Cle-caspase-3 at 14D onward. Cle-caspase-3-positive cells were highly expressed both in seminiferous tubule and in interstitial tissues at 21D and 28D. Furthermore, Cle-caspase-3 was detected in some peritubular myoid cells of atrophic tubules at 21D and 28D. The data of the graph supported that percentage expression of Cle-caspase-3 per tubule increased significantly at 21D (5.078 ± 0.161) and 28D (8.021 ± 0.097) when compared with control (3.426 ± 0.228; Fig. 8).

Figure 8.

Fluorescence images of mouse seminiferous tubules showed the expression of cleaved Caspase-3 (Cle-caspase-3; red) in control group (0D) and cryptorchidism groups (3D, 5D, 7D, 14D, 21D, and 28D). Nuclei were counterstained with Hoechst (blue). Cle-caspase-3 expressed slightly in basal compartment of tubule at 3D, the same as control. Strong expression of Cle-caspase-3 in germ cell started to be revealed at 5D. At 7D, Cle-caspase-3 was detected in germ cell, spermatocytes, and spermatids. Cells in the seminiferous tubule had expression of Cle-caspase-3, as did cells in interstitial tissue at 14D, 21D, and 28D. Cle-caspase-3-positive cell count was higher in both seminiferous tubule and interstitial tissue at 21D and 28D, compared with control. The graph showed that the percentage expression of Cle-caspase-3 per tubule increased significantly at 21D and 28D when compared with control. *p<0.05. (Scale bars = 50 micrometers.)

Discussion

In this study, we intended to elucidate the effect of higher abdominal temperature as it disrupted the mechanism of spermatogenesis, resulting in morphological abnormality of cryptorchid testes and abnormal expression of protein markers in spermatogenic cells and supporting Sertoli cells. We studied this using experimental cryptorchidism model mice at days 3, 5, 7, 14, 21, and 28 post-operatively to show the timeline of abnormality and explain changes in the testis.

The normal cycle of mouse spermatogenesis is around 8.6 days, and the appropriate time required for the differentiation process from A spermatogonia developing into mature sperm is about 35 days.^25,26 Previous studies reported that the H&E staining cryptorchid testes of rats presented a sharp decrease of germ cells and multinucleated giant cells that appeared at 3D post-operatively, appearing maximally at 5D. ³ Other studies showed that surgically induced cryptorchidism in mice deceased testicular weight at 4D post-operatively, and the seminiferous tubule became atrophic at 20D.^1,27 Our results revealed the typical morphology of cryptorchid testis at 3D and revealed increasing morphological abnormality at 5D. Light microscope images of cryptorchid testis in 5D group revealed detached germ cells in the lumen of the seminiferous tubule and some spermatids had developed a small vacuole close to their nuclei, whereas some seminiferous tubules showed abnormal formation of the acrosome. Abnormal formation of acrosomes in Step 6 and the presence of multinucleated giant cells, resulting from spermatogonia, were also detected by TEM at 5D. The high abdominal temperature also induced abnormal formation of sperm flagellum. Ac-tubulin was seen as a short tubular pattern at the original site of axoneme. Abnormal development of spermatid both in acrosomal formation and in flagellum elongation was involved with Ac-tubulin expression. During acrosomal biogenesis, non-acrosomal vesicles are subsequently transported by molecular motors that rely on microtubules of the manchette. This transport occurs toward both the acrosomal region and the developing spermatid tail. ¹⁴ Recent discoveries have revealed that HOOK2 plays a vital role in preserving the structure of centrioles and facilitating the assembly of primary cilia, including sperm flagellum. ²⁸ Ac-tubulin, serving as an axoneme marker, exhibits colocalization with HOOK2 on the microtubules of the manchette. ¹⁷ Likewise, the decreasing expression of Cadm1was initially observed in the caudal portion of elongated spermatid. In addition, some Sertoli cells revealed endoplasmic reticulum swelling at 5D.

The elongating spermatid was detected in the cytoplasm of Sertoli cell by H&E staining and confirmed by TEM at 7D and 14D. This could be caused by the loss function of Sertoli cells to support spermatogonia development. Moreover, Sertoli cells also showed swollen mitochondria in their cytoplasm. There are many studies that have reported that the higher temperature induced the intrinsic apoptotic pathway in mitochondria and affected the morphology and function of Sertoli cells.^29–31 Also, the high abdominal temperature affected the cytoskeleton of Sertoli cells in the cryptorchid testes of monkeys. The expression of vimentin in the cytoplasm of Sertoli cells increased, reducing the reported number of germ cells. ³⁰ Microtubules are predominately cytoskeleton in the apical part and arranged in the long axis of the Sertoli cell. ³² Microtubules play vital roles in providing structural support to the seminiferous epithelium, facilitating the movement of spermatogenic cells, and enabling the release of elongating spermatids into the tubule’s lumen. Within Sertoli cells, the microtubules are arranged from the nucleus to the apical cytoplasmic processes, and they also align with ES junctions connecting the Sertoli cell and elongating spermatid. ³³ In addition, the Ac-tubulin contributes to the differentiation of microtubule structure and function. IF staining of Ac-tubulin was strongly expressed in the axoneme and the apical part of Sertoli cells. Our results showed that the expression of Ac-tubulin in cytoplasm of Sertoli cells remained in cryptorchid testes but that the arrangement of acetylates tubulin at the apical compartment was changed. So, we can assume that the pattern change of protein expression leads to the disability of Sertoli cell to release elongating spermatids. Sertoli cells expressed Nectin-2 in the plasma membrane, both in the apical and in the basal parts. Nectin-2 was present at the Sertoli–Sertoli junctions of the basal ectoplasmic region and interacted with Nectin-3 at the Sertoli–Spermatid junctions of the apical ectoplasmic region. ⁹ We observed the expression of Nectin-2 and found that Nectin-2 activity decreased at the Sertoli–spermatid junction and decreased significantly at 21D and 28D in the cryptorchid testis. This result supports the loss of function of the apical part of the Sertoli cells. Meanwhile, the expression of WT1 in nuclei of Sertoli cells showed no statistical difference between control and cryptorchidism groups, but the number of WT1-positive cells per tubule increased slightly at 28D. Moreover, BrdU was not expressed in the Sertoli cells because the mature Sertoli cells in the control and cryptorchid testes were found to be non-proliferative cells. These all confirm the evidence of failure of the Sertoli cells in moving elongating spermatids to the apical compartment and failure of release into the tubular lumen and resultant phagocytosis.

Histopathology of the cryptorchid testis revealed the irregular shape and vacuole development in the head of elongating spermatids at 14D. This result corresponds to the expression of Nectin-3 that decreased slightly at 14D and significantly at 21D and 28D. Moreover, the irregular shapes of nuclei of sperm head in Nectin-3^−/− mice were also confirmed by electron microscope image. ¹⁰ At 14D, Ac-tubulin disappeared in the axoneme and was shown as a circular pattern in some spermatids. In addition, Ac-tubulin was detected in giant cells at Stage XII of seminiferous tubule of the 21D group and was identified in the cytoplasm of Sertoli cells at 28D. Our results have shown that high abdominal temperature disrupts the normal expression of Ac-tubulin in both spermatids and supporting Sertoli cells. Abnormal development of spermatids can be observed starting at 5D, as indicated by the abnormal expression of Ac-tubulin. Furthermore, the high abdominal temperature can affect the function of the cytoskeleton in Sertoli cells, causing damage and altering the pattern of protein alignment, which ultimately leads to the sloughing of the seminiferous epithelium. By 28D, the cryptorchid testis experienced a complete loss of elongating spermatids. However, Ac-tubulin was still identified in the cytoplasm of Sertoli cells and the seminiferous epithelium. The expression of Ac-tubulin observed in seminiferous epithelium suggesting its presence in remaining round spermatids. Cle-caspase-3-positive cells were highly expressed, both in the seminiferous tubule and in the interstitial tissue, at 21D and 28D. The ultrastructure of cryptorchid testis revealed swollen mitochondria, a lot of multivesicular bodies, and micro-autophagy in both Sertoli cells and other degenerating cells, leading to apoptosis. A recent study reported that autophagy, apoptosis, and oxidative stress in the testis are a response to heat stress. Autophagosomes could be observed by TEM at 24 hr after the lower parts of the mice’s body was submerged in a thermostatically controlled water bath for 20 min in 39C. Spermatocytes had the highest positive response to TUNEL staining at 24 hr, after induction at 42C. Malondialdehyde and hydrogen peroxide are two markers for reactive oxygen species that are also increased after heat treatment at 42C. ³⁴ In addition, we found the presence of irregular collagen fibers in the basal laminar and also lysosome-bound nanoparticles in the peritubular myoid cells of the cryptorchid testes. We can confirm Cle-caspase-3-positive apoptosis of peritubular myoid cells at 21D and 28D.

We found that development of multinucleated giant cells, resulting from spermatogonia, spermatocytes, and round and elongating spermatids, depended on the number of days post-operatively. Germ cells are sensitive to heat stimulation due to their high mitotic activity ³⁵ and resultant DNA damage to the primary spermatocytes and round spermatids can be examined by TUNEL assay. ³⁶ The S phase in the germ cells is the most significant step in the DNA replication to produce the genetic material during the cell cycle. DNA polymerases are the essential enzymes for DNA replication. The denaturation and dysfunction of DNA polymerases would be affected by heat stress that induced double-strand breaks in DNA. ³⁵ The effect of a high temperature of 37C decreased the expressions and activities of testicular DNA polymerases α, β, and γ in an in vitro study. ³⁷ Multinucleated giant cells form, resulting from damaged germ cells, due to the disappearance of the intercellular bridge. Generally, cytoplasmic sharing occurs after the process of meiosis. The mRNA and organelles move through the intercellular bridge to permit the cytoplasmic sharing between haploid spermatids that is necessary for haploid germ cells to remain phenotypically diploid.^38–40 The high abdominal temperature disrupted this cytoplasmic sharing, delayed development, and led germ cells to fuse into one large cell. Furthermore, the degeneration of spermatocytes or spermatids and expansion of the intercellular bridges induced the formation of multinucleated giant cells. ⁴¹ Both degenerating spermatogenic cells and apoptotic cells are involved in the formation of multinucleated giant cells. These giant multinucleated apoptotic cells were visualized in the cryptorchid testis at 28D. The cytoplasm revealed autophagy and a lot of swollen mitochondria. The apoptotic cells underwent nuclear condensation with vacuolization and then fused together to share the cell membrane. A previous study reported the role of autophagy-related gene 7, in initiation and performance of autophagy, and suggested that autophagy acts as a partner of apoptosis, to induce cell death and disrupt spermatogenesis after heat stress. ⁴²

Our study showed that the multinucleated giant cells did not express Cadm1 and Nectin-2 but presented Nectin-3 expression in the cryptorchid testis before 21D. Cadm1 is involved in cell–cell interaction at the plasma membrane level, between spermatogenic cells (intermediate spermatogonia to early pachytene, spermatocytes Steps 7–16 spermatids, and caudal portion of elongating spermatids) and PVR on a Sertoli cell. ⁸ The high abdominal temperature affects the organelles and cytoskeleton in Sertoli cell’s cytoplasm causing loss of the receptor’s function on the plasma membrane. In the same way, germ cells, which were injured by the heat stimulation and decreased expression of Cadm1, lost the cell–cell interaction and fused their membranes to form giant cells. The significantly decreased Cadm1 expression at 14D and the increase of Cle-caspase-3 expression at 21D and 28D can be used to explain why Cadm1 expression in the giant cells was not detected. There are many studies reporting that the high temperature induced the intrinsic apoptotic pathway in mitochondria and affected the morphology and function of Sertoli cells.^29–31 We observed the expression of Nectin-2 and found that Nectin-2 decreased in the Sertoli–spermatid junction and decreased significantly at 21D and 28D in the cryptorchid testis. This result supports the loss of function of the apical part of the Sertoli cells. Moreover, the abnormal Sertoli cells had not been found in the multinucleated giant cell when observed by TEM. This result supports the absence of Nectin-2 expression in the giant cell. A previous study demonstrated the localization and interaction of Nectin-2 and Nectin-3 in mouse testis at the junctions between the membranes of Sertoli cells and elongating (Steps 8–16) spermatids, respectively. ¹² The elongating spermatid development was delayed more by the heat stimulation compared with germ cells and round spermatids. ³⁶ We observed the histopathology of cryptorchid testis and found that 7D group still represented all spermatogenic cells and supporting Sertoli cells but lost the elongating spermatids at 21D and 28D. This result correlated with Nectin-3 expression in the giant multinucleated elongating spermatid cell at 7D. When spermatids are released from Sertoli cells as spermatozoa, Nectin-3 still expresses in the head portion of spermatozoa, at the same time Nectin-2 can be recycled by the endosome.^43,44 Nectin-3 has an antiapoptotic reaction and prevents programmed cell death by regulating the activation of the phosphatidylinositol 3-kinase–Akt signaling pathway for cell survival.^43,45 Accordingly, the multinucleated giant cells resulting from round spermatid expressed Nectin-3 due to the extent of DNA damage in round spermatids due to heat stimulation. However, it is still unknown why some round spermatids underwent translocation of Nectin-3 expression from their plasma membrane into their nuclei.

The caput of epididymis is the first area of sperm storage after transfer from the testis. The immature sperm leave the testis and enter the caput epididymis. Sperm develop to maturity during epididymal transition from the caput to the caudal part of the epididymis. The number of sperm in the caput of epididymis decreased gradually post-operatively in the cryptorchidism model. In contrast, the epididymis of 14D, 21D, and 28D groups had increased numbers of detached germ cells, giant cells, and debris in the lumen. This was confirmed by a study in mice exposed to a higher temperature of 37–38C for 8 hr 3 days continuously. The percentage of motile spermatozoa in caudal epididymis reduced significantly in the heat-treated mice. ³⁸ Our result confirmed the reduction of the weight of cryptorchid testes in 21D and 28D significantly when compared with the control left testis and self-control right testis. In contrast, the epididymal weight showed no different statistical significance. Similarly, the previous study reported that the size of left cryptorchid testis was reduced compared to the self-control testis at days 7, 14, 21, and 28 post-operation and also was reduced when compared to the left testes of the sham-operated group. Nevertheless, the size of epididymis showed no obvious change. ²³ In addition, the weights of epididymis in unilateral cryptorchidism mice were not altered by both 15 and 30 days of study. ² However, the re-location of the cryptorchid testis might involve a degree of damage. A previous review of the medical records in patients with unilateral cryptorchidism reported an association of hormone level with pretreatment testicular location. The patients with inguinal cryptorchidism had a significantly lower level of inhibin B, patients with the ectopic testis had a higher level of luteinizing hormone than other locations, and intra-abdominal testes had a slightly increased risk for infertility. ³⁶ In contrast, the current review of testicular location is limited by the small number of cryptorchidism patients, which possibly affects the statistical power and so does not support the idea of higher location being associated with inadequate hormonal function. ³⁷ Even so, our study found that the size of intra-abdominal cryptorchid testes was smaller than pelvic cryptorchid testes and the testicular weight also decreased by around 50%, whereas the weight of pelvic testes did not significantly alter when compared with control testes.

In conclusion, our results show that cryptorchidism’s duration time relates directly to the development of morphological abnormality. The abnormal expression of protein markers in spermatogenic and Sertoli cells explains how spermatogenesis is affected by high abdominal temperature. Not only the cells in the seminiferous tubule were damaged by heat stimulation, but peritubular myoid cells and Leydig cells were also affected. This result can be explained by the abnormal changes observed in testis, encompassing both cell morphology and protein expression, which play crucial roles in spermatogenesis. In humans, several factors contribute to testicular descent during the development of the male reproductive system, such as hormonal regulation of insulin-like hormone 3, anti-Müllerian hormone, androgens, and genetic mutations. However, it is important to note that the current study focuses on surgically induced cryptorchidism in animal models, which serve as a disease model in adult normal mice. Therefore, the data presented in this study primarily serve as a laboratory experiment and do not specifically address the impact of hormonal control and gene expression function.

Appendix

List of primary and secondary antibodies for immunofluorescence used to identify protein markers in control and cryptorchid testes.

Appendix Table 1.

Primary and Secondary Antibodies for IF Used in This Study.

Antibody	Target	Code No., Vendor	Origin (Clone No.)	Working Dilution	Cross-Reactivity with Any Proteins in Testis
Primary	Polyclonal anti-Cadm1	In house production46,47	Rabbit	1:400	No7,8,48
Primary	Monoclonal anti-Nectin-2	ab16912, Abcam	Rat	1:200	No 13
Primary	Polyclonal anti-Nectin-3	ab63931, Abcam	Rabbit	1:200	No13,49,50
Primary	Monoclonal anti-acetylated tubulin	T7451, Sigma-Aldrich	Mouse	1:500	No51–53
Primary	Recombinant monoclonal anti-WT1	ab89901, Abcam	Rabbit	1:200	No54–57
Primary	Monoclonal anti-BrdU	B8434, Sigma-Aldrich	Mouse	1:150	No7,58
Primary	Polyclonal anti-vimentin	ab45939, Abcam	Rabbit	1:1000	No59,60
Secondary	Anti-rabbit IgG conjugated to Alexa Fluor 594 (red)	ab150080, Abcam	Goat	1:400	—
Secondary	Anti-rabbit IgG conjugated to Alexa Fluor 488 (green)	ab150077, Abcam	Goat	1:400	—
Secondary	Anti-rat IgG conjugated to Alexa Fluor 488 (green)	ab150157, Abcam	Goat	1:400	—
Secondary	Anti-mouse IgG conjugated to Alexa Fluor 488 (green)	ab150113, Abcam	Mouse	1:400	—
Secondary	Anti-mouse IgG conjugated to Alexa Fluor 594 (red)	ab150116, Abcam	Goat	1:400	—

Samples were fixed in 4% paraformaldehyde.

Abbreviations: IgG, immunoglobulin; IF, immunofluorescence.