Heme oxygenase 1 (HO1) regulates autophagy and apoptosis via the PI3K/AKT/mTOR signaling pathway of yak Sertoli cells.

Successful male reproduction depends on healthy testes. Autophagy has been confirmed to be active during many cellular events associated with the testes. It is not only crucial for testicular spermatogenesis but is also an essential regulatory mechanism for Sertoli cell (SCs) ectoplasmic specialization integrity and normal function of the blood-testis-barrier. Hypoxic stress induces oxidative damage, apoptosis, and autophagy, negatively affecting the male reproductive system. Cryptorchidism is a common condition associated with infertility. Recent studies have demonstrated that hypoxia-induced miRNAs and their transcription factors are highly expressed in the testicular tissue of infertile patients. Heme oxygenase 1 (HO1) is a heat-shock protein family member associated with cellular antioxidant defense and anti-apoptotic functions. The present study found that the HO1 mRNA and protein are up-regulated in yak cryptorchidism compared to normal testes. Next, we investigated the expression of HO1 in the SCs exposed to hypoxic stress and characterized the expression of key molecules involved in autophagy and apoptosis. The results showed that hypoxic stress induced the upregulation of autophagy of SCs. The down-regulation of HO1 using siRNA increases autophagy and decreases apoptosis, while the over-expression of HO1 attenuates autophagy and increases apoptosis. Furthermore, HO1 regulates autophagy and apoptosis via the PI3K/AKT/mTOR signaling pathway. These results will be helpful for further understanding the regulatory mechanisms of HO1 in yak cryptorchidism.

Testicular expression of heat SHOCK proteins 60, 70, and 90 in cryptorchid horses.

Heat shock proteins are the most evolutionarily conserved protein families induced by stressors including hyperthermia. In the context of pathologies of the male reproductive tract, cryptorchidism is the most common genital defect that compromises the reproductive potential of the male because it induces an increase in intratesticular temperature. In equine species, cryptorchidism affects almost 9 % of newborns and few studies have been carried out on the molecular aspects of the retained testis. In this study, the expression pattern of HSP60, 70, and 90 in abdominal and inguinal testes, in their contralateral descended normally testes, and in testes of normal horses were investigated by Western blot and immunohistochemistry. The histomorphological investigation of retained and scrotal testes was also investigated. The seminiferous epithelium of the retained testes showed a vacuolized appearance and displayed a completely blocked spermatogenesis for lacking meiotic and spermiogenetic cells. On the contrary, the contralateral scrotal testes did not show morphological damage and the seminiferous epithelium displayed all phases of the spermatogenetic cycle as in the normal testes. The morphology of Leydig cells was not affected by the cryptorchid state. Western blot and immunohistochemistry evidenced that equine testis (both scrotal and retained) expresses the three investigated HSPs. More in detail, the Western blot evidenced that HSP70 is the more expressed chaperone and that together with HSP90 it is highly expressed in the retained gonad (P < 0.05). The immunohistochemistry revealed the presence of the three HSPs in the spermatogonia of normal and cryptorchid testes. Spermatogonia of retained testes showed the lowest expression of HSP60 and the highest expression of HSP90. Spermatocytes, spermatids of scrotal testes, and the Sertoli cells of retained and scrotal testes did not display HSP60 whereas expressed HSP70 and HSP90. These two proteins were also localized in the nucleus of the premeiotic cells. The Leydig cells displayed the three HSPs with the higher immunostaining of HSP70 and 90 in the cryptorchid testes. The results indicate that the heat stress condition occurring in the cryptorchid testis influences the expression of HSPs.

Histological study of Bactrian camel cryptorchidism and expression of immunoglobulin λ light chain in the testicular and epididymis of cryptorchid Bactrian camel.

Lambda light chains (λ-LCs) are frequently responsible for triggering the activation of inflammatory factors in autoimmune disorders, and an increase in their levels will cause various pathological changes in serum. The aim of this study was to determine the histological differences between the epididymis and testis of normal and cryptorchid Bactrian camels and the differences in λ-LC expression in the epididymis and testis of normal and cryptorchid Bactrian camels. Haematoxylin and eosin (H&E) staining was used to examine the pathological changes in cryptorchidism. The gene and protein levels of λ-LC were determined using RT-qPCR and western blot. The distribution of λ-LCs was assessed by immunohistochemistry and immunofluorescence. Compared with that in normal Bactrian camels, the diameter of the epididymal lumen and the thickness of the epithelium were decreased in the epididymis of cryptorchidic animals. Additionally, no sperm was detected in the cavity of the cryptorchidic epididymis. Meanwhile, the expression of λ-LC was significantly increased in the cryptorchidic epididymis at both the mRNA and protein levels (p < .05). The highest protein expression of λ-LC was found in epididymal epithelial halo cells and testicular Sertoli cells. These findings suggested that the structural changes observed in the epididymal epithelium of cryptorchidic camels affect its secretory and absorptive functions. Additionally, the high level of λ-LC expression recorded in halo cells suggested that these cells play an important role in epithelial immunity in cryptorchidic Bactrian camels. Furthermore, the high λ-LC expression levels detected in normal testicular Sertoli cells indicated that λ-LCs may be involved in spermatogenesis. The results of this study provide clues for an in-depth study of immunological sterility in cryptorchidic Bactrian camels.

MicroRNA as epigenetic regulators of canine cryptorchidism.

Cryptorchidism, the failed descent of one or both testes into the scrotum, is a common developmental disorder in male dogs. Cryptorchidism may affect canine fertility, reducing the quality of the semen, and may promote spermatic cord torsion and onset of neoplasia. MicroRNAs (miRNAs) are epigenetic regulators of gene expression and their dysregulation is associated with disorders of spermatogenesis and testis neoplasia. The present study aimed at investigating the expression of miRNAs in formalin-fixed, paraffin-embedded (FFPE) canine retained testes and testes affected by seminoma, and at integrating miRNAs to their target genes. Forty testicular FFPE specimens from 30 dogs were included - 10 scrotal and 10 contralateral retained from 10 unilateral cryptorchid dogs; 10 tumoral testes affected by seminoma from non-cryptorchid dogs; 10 scrotal normal testes from non-cryptorchid dogs included as the control. The expression level of three miRNAs, namely miR-302c-3p, miR-302a-3p, and miR-371-3p, associated with testicular disorders, were quantified using RT-qPCR. The comparative analysis demonstrated that the level of miR-302a-3p and miR-371a-3p were quantifiable exclusively in control testes. The expression level of miR-302c-3p was higher in the control than in the other groups; its expression decreased in retained testes compared to scrotal testes and testes with seminoma. Gene Ontology analysis pointed out that these miRNAs may be involved in the modulation of estrogen and thyroid hormone signaling pathways. In conclusion, this study demonstrated that miRNAs are dysregulated in canine cryptorchid and seminoma-affected testes compared to control tissues, confirming the pivotal role of miRNAs in cryptorchidism.

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

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.

Transcriptomic Analysis of Testicular Gene Expression in a Dog Model of Experimentally Induced Cryptorchidism.

Cryptorchidism, a condition in which testes fail to descend from the abdomen into the scrotum, is a risk factor for infertility and germ cell cancer. Normally, tight junctions between adjacent Sertoli cells in the testes form a blood-testes barrier that regulates spermatogenesis; however, the effect of cryptorchidism on tight junctions is not well-understood. We established a model of heat-induced testicular damage in dogs using surgical cryptorchidism. We sequenced RNA to investigate whether certain transcripts are expressed at higher rates in heat-damaged versus normally descended testes. Claudins, cell adhesion molecules, were relatively highly expressed in cryptorchid testes: claudins 2, 3, 5, 11, and 18 were significantly increased in cryptorchid testes and reduced by orchiopexy. SOX9-positive Sertoli cells were present in the seminiferous tubules in both cryptorchid and control testes. Using real-time PCR and Western blot analysis to compare Sertoli cells cultured at 34 °C and 37 °C, we found that Sertoli cell claudins 2, 3, 5, 11, and 18 were significantly increased at 37 °C; however, accumulation was higher in the G0/G1 phase in Sertoli cells cultured at 34 °C. These results indicate that testicular hyperthermia caused by cryptorchidism affects claudin expression, regulated germ cell death, and the proliferation of Sertoli cells.

Organotypic Culture of Testicular Tissue from Infant Boys with Cryptorchidism.

Organotypic culture of human fetal testis has achieved fertilization-competent spermatids followed by blastocysts development. This study focuses on whether the organotypic culture of testicular tissue from infant boys with cryptorchidism could support the development of spermatogonia and somatic cells. Frozen-thawed tissues were cultured in two different media, with or without retinoic acid (RA), for 60 days and evaluated by tissue morphology and immunostaining using germ and somatic cell markers. During the 60-day culture, spermatocytes stained by boule-like RNA-binding protein (BOLL) were induced in biopsies cultured with RA. Increased AR expression (p < 0.001) and decreased AMH expression (p < 0.001) in Sertoli cells indicated advancement of Sertoli cell maturity. An increased number of SOX9-positive Sertoli cells (p < 0.05) was observed, while the percentage of tubules with spermatogonia was reduced (p < 0.001). More tubules with alpha-smooth muscle actin (ACTA, peritubular myoid cells (PTMCs) marker) were observed in an RA-absent medium (p = 0.02). CYP17A1/STAR-positive Leydig cells demonstrated sustained steroidogenic function. Our culture conditions support the initiation of spermatocytes and enhanced maturation of Sertoli cells and PTMCs within infant testicular tissues. This study may be a basis for future studies focusing on maintaining and increasing the number of spermatogonia and identifying different factors and hormones, further advancing in vitro spermatogenesis.

The effects and molecular mechanism of heat stress on spermatogenesis and the mitigation measures.

Under normal conditions, to achieve optimal spermatogenesis, the temperature of the testes should be 2-6 °C lower than body temperature. Cryptorchidism is one of the common pathogenic factors of male infertility. The increase of testicular temperature in male cryptorchidism patients leads to the disorder of body regulation and balance, induces the oxidative stress response of germ cells, destroys the integrity of sperm DNA, yields morphologically abnormal sperm, and leads to excessive apoptosis of germ cells. These physiological changes in the body can reduce sperm fertility and lead to male infertility. This paper describes the factors causing testicular heat stress, including lifestyle and behavioral factors, occupational and environmental factors (external factors), and clinical factors caused by pathological conditions (internal factors). Studies have shown that wearing tight pants or an inappropriate posture when sitting for a long time in daily life, and an increase in ambient temperature caused by different seasons or in different areas, can cause an increase in testicular temperature, induces testicular oxidative stress response, and reduce male fertility. The occurrence of cryptorchidism causes pathological changes within the testis and sperm, such as increased germ cell apoptosis, DNA damage in sperm cells, changes in gene expression, increase in chromosome aneuploidy, and changes in Na+/K+-ATPase activity, etc. At the end of the article, we list some substances that can relieve oxidative stress in tissues, such as trigonelline, melatonin, R. apetalus, and angelica powder. These substances can protect testicular tissue and relieve the damage caused by excessive oxidative stress.

The inhibition of CFTR in the descended testis of SD rats with unilateral cryptorchidism induced by di-(2-ethylhexyl) phthalate (DEHP).

Di-(2-ethylhexyl) phthalate (DEHP) is a kind of environmental endocrine disruptors (EEDs), which has been confirmed to cause serious consequences, such as cryptorchidism. Patients with unilateral cryptorchidism still had oligospermia or infertility even if they received orchidopexy before puberty. Testicular dysgenesis syndrome (TDS) attributes this kind of problems to the abnormal testicular development during the embryonic period, and considers that the environmental exposure factors during pregnancy play a major role. Therefore, for unilateral cryptorchidism, even if one testicle has dropped to scrotum, it may be exposed to these substances and cause damage. Cystic fibrosis transmembrane conduction regulator (CFTR) is very important for the maturation of male reproductive system. Previously, cryptorchidism was thought to cause abnormal expression of heat sensitive protein CFTR in testis, but the expression of CFTR in healthy side (descended side) testis was not clear. In this study, we established SD rats with unilateral cryptorchidism by exposure to DEHP (500 mg/kg/day) during pregnancy, and detected the expression of CFTR and downstream signal NF-κB/COX-2/PGE2 in bilateral testis. Finally, we found that the expression of CFTR and downstream signal NF-κB/COX-2/PGE2 in the undescended testis was significantly abnormal, but the expression of them in the descended testis was also abnormal to some extent. Therefore, we speculate that in addition to high temperature will affect the expression of CFTR, there may be other factors that cause abnormal expression of CFTR induced by DEHP, and lead to abnormal male reproductive function eventually, but the specific mechanism needs to be further studied.

Comparative transcriptome analysis of experimental cryptorchidism: of mice and cynomolgus monkeys.

In most mammalian species, the testis descends from the abdomen into the scrotum during fetal or neonatal life. The failure of testicular descent, a pathological condition known as cryptorchidism, has long been the subject of scientific interest in a wide range of fields, including medicine, developmental biology, and evolutionary biology. In this study, we analyzed global gene expression changes associated with experimental cryptorchidism in mice by using RNA-seq. A total of 453 differentially expressed genes were identified, of which 236 genes were upregulated, and 217 genes were downregulated. Gene ontology, pathway, and gene network analysis highlighted the activation of inflammatory response in experimental cryptorchidism. By examining the promoter regions of differentially expressed genes, we identified 12 causal transcription factors. In addition, we also induced experimental cryptorchidism in two cynomolgus monkeys and performed RNA-seq. A cross-species comparison was performed at the gene expression level. Our study provides a valuable resource for further understanding the molecular mechanisms of cryptorchidism in mammals.

Low Serum Inhibin B/Follicle-Stimulating Hormones and Anti-Müllerian Hormone/Follicle-Stimulating Hormones Ratios as Markers of Decreased Germ Cells in Infants with Bilateral Cryptorchidism.

PURPOSE: In cryptorchidism, germ cell development failure presents from infancy and may be reflected by altered hormonal levels produced by Sertoli cells. Our object was to assess for associations between serum hormone levels and testicular histopathology in cryptorchidism with an infertility risk according to the pretreatment undescended testicular positions. MATERIALS AND METHODS: Prepubertal cryptorchid boys aged 7-91 (median 20) months who underwent orchidopexy between 2014 and 2019 were included (122 unilateral [median 19 months {range 7-91}], 23 bilateral [24 months {11-81}]). We evaluated the pretreatment testicular position and size; serum hormone levels; and the mean number of germ cells per tubule transverse section (G/T). We also performed a subgroup analysis of boys aged ≤24 months at orchidopexy. RESULTS: Serum inhibin B levels and G/T were significantly lower in bilateral than in unilateral cryptorchid boys (median 96 [range 46-197] pg/ml vs 125 [21-354] pg/ml, p=0.026; 0.20 [0-2.59] vs 0.65 [0-4.55], p <0.001, respectively). Inhibin B/follicle-stimulating hormones (FSH) and anti-Müllerian hormone (AMH)/FSH ratios were positively correlated with G/T in bilateral cryptorchid boys aged ≤24 months (12, p=0.008 and p=0.019, respectively). Low inhibin B/FSH and AMH/FSH ratios and high FSH were predictors of impaired G/T as per receiver operating characteristic curves (p=0.019, p=0.004 and p=0.004, respectively), whereas in unilateral cryptorchid boys aged ≤24 months, serum hormone levels and G/T did not differ with the pretreatment testicular positions. CONCLUSIONS: In bilateral cryptorchid boys aged ≤24 months at orchidopexy, low inhibin B/FSH and AMH/FSH ratios may reflect impaired G/T and future infertility risk.

The G-Protein-Coupled Membrane Estrogen Receptor Is Present in Horse Cryptorchid Testes and Mediates Downstream Pathways.

Cryptorchidism in horses is a commonly occurring malformation. The molecular basis of this pathology is not fully known. In addition, the origins of high intratesticular estrogen levels in horses remain obscure. In order to investigate the role of the G-protein-coupled membrane estrogen receptor (GPER) and establish histological and biochemical cryptorchid testis status, healthy and cryptorchid horse testes were subjected to scanning electron microscopy analysis, histochemical staining for total protein (with naphthol blue black; NBB), acid content (with toluidine blue O; TBO), and polysaccharide content (with periodic acid-Schiff; PAS). The expression of GPER was analyzed by immunohistochemistry and Western blot. GPER-mediated intracellular cAMP and calcium (Ca2+) signaling were measured immunoenzymatically or colorimetrically. Our data revealed changes in the distribution of polysaccharide content but not the protein and acid content in the cryptorchid testis. Polysaccharides seemed to be partially translocated from the interstitial compartment to the seminiferous tubule compartment. Moreover, the markedly decreased expression of GPER and GPER downstream molecules, cAMP and Ca2+, suggests their potential role in testis pathology. Increased estrogen levels in cryptorchid conditions may be linked to disturbed GPER signaling. We postulate that GPER is a prominent key player in testis development and function and may be used as a new biomarker of horse testis in health and disease.

Experimental Cryptorchidism Causes Chronic Inflammation and a Progressive Decline in Sertoli Cell and Leydig Cell Function in the Adult Rat Testis.

Cryptorchidism causes spermatogenic failure and reduced serum androgen levels, as well as testicular oedema and fibrosis, which are hallmarks of inflammation. However, the role of inflammation and the effects of cryptorchidism on Sertoli cell and Leydig cell function at the molecular level remain ill-defined. Bilateral cryptorchidism was surgically induced in adult rats for 7 and 14 weeks. Testis weights decreased to 40% of normal within 7 weeks, due to loss of all developing spermatogenic cells except spermatogonia, but did not decrease further at 14 weeks. Serum FSH and LH were increased at both time points, consistent with a loss of feedback by inhibin and testosterone. This damage was accompanied by progressive accumulation of interstitial fluid and peritubular fibrosis, and a progressive decline of several critical Sertoli cell genes (Sox9, Inha (inhbin α-subunit), Cldn11 (claudin 11), Gja1 (connexin 43), and Il1a (interleukin-1α)) and the Leydig cell steroidogenic enzymes, Cyp11a1, Hsd3b1, and Hs17b3. Activin B and the activin-binding protein, follistatin, also declined, but the intratesticular concentration of activin A, which is a regulator of inflammatory responses, was largely unaffected at either time point. Expression of genes involved in inflammation (Tnf, Il10, Il1b, Mcp1) and fibrosis (Acta2, Col1a1) were considerably elevated at both time points. These data indicate that induction of experimental cryptorchidism, which causes complete failure of spermatogenesis in the adult rat, also induces chronic testicular inflammation, manifesting in oedema and fibrosis, and a progressive decline of Sertoli and Leydig cell gene expression and function.

Differential abundances of AQP3 and AQP5 in reproductive tissues from dogs with and without cryptorchidism.

Aquaporins (AQPs) are integral transmembrane proteins facilitating transport of water and small solutes, such as glycerol and urea, between cells. In male reproductive tracts, AQPs maintain a milieu conducive for sperm formation, maturation, and storage. The aim of this study was to clarify effects of testicular and epidydimal function on male fertility by investigating localisation and abundances of AQP3 and AQP5 in testes and epididymal segments from dogs with and without unilateral cryptorchidism. Immunohistochemistry results indicated AQP3 and AQP5 have different distribution patterns in reproductive tissues of dogs with and without unilateral cryptorchidism. The AQP3, an aquaglyceroprotein, is present in different germ and Sertoli cells in testis of dogs without cryptorchidism. The AQP5 protein was not detected in germ cells but was present in Sertoli and Leydig cells and in endothelia of blood vessels. In cryptorchid dogs, AQP3 was detected in early-developing germ and Sertoli cells, and AQP5 had a distribution pattern similar to testes of dogs without cryptorchidism. In the epididymis, AQP3 and AQP5 were localised in epithelial cells of dogs with and without cryptorchidism in a cell-specific manner. The AQP3 and AQP5 protein was in larger abundance in the gonads from dogs with and without cryptorchidism. In contrast, AQP3 and AQP5 abundance increased in each segment of the cryptorchid epididymis, likely as a compensatory mechanism associated with the pathologic condition. These results indicate involvement of AQP3 and AQP5 in spermatogenesis and sperm maturation. Results from the present study indicate dogs are a useful for comparative reproductive biology studies.

INSL-3 protein expression in normal and cryptorchid testes of Ziwuling black goats.

Ziwuling black goats are typically found in loess plateaus regions and the Ziwuling Nature Reserve. Cryptorchidism is a common disease in this inbred goat, and its pathogenesis has been linked with the expression of insulin-like factor 3 (INSL-3). Therefore, this study aimed to investigate anatomical alterations caused by cryptorchism and the expression and distribution of INSL-3 in normal and cryptorchid testicular tissues. The testicular tissues of 6-month-old Ziwuling black goats were collected for microscopic analyses using histochemical, immunohistochemical, immunofluorescence and biometrical methods, as well as Western blotting to compare the expression and distribution of INSL-3. A lower expression of INSL-3 was observed in cryptorchid compared with normal testicular tissues (p < .01). Cryptorchidism caused a significant reduction in layers of spermatogenic epithelium and tubule areas in Ziwuling black goat (p < .01). The interstitial to seminiferous tubule area ratio was larger in cryptorchid than in normal group. Periodic Acid-Schiff (PAS) staining revealed pronounced positive bands in the interstitial tissue, while positive Alcian blue (AB) staining was not clear, and AB-PAS staining revealed a positive red band in the basement membrane of cryptorchid group. Immunofluorescence revealed a strong signal of INSL-3 expression in Sertoli and peritubular myoid cells, and moderate signal in Leydig and spermatogenic cells in the normal group. However, in cryptorchid testicular tissues, the signal of INSL-3 expression was strong in primary spermatocytes, occasional in Sertoli cells, limited in Leydig cells and absent in peritubular myoid cells. Furthermore, immunohistochemistry showed that INSL-3 expression was higher in normal testes compared with cryptorchid testicular tissues (p < .05), especially in primary spermatocytes and Sertoli cells. Collectively, our results indicate that cryptorchidism is closely related to the disorder of acid glycoprotein metabolism and the reduction in release of INSL-3 from Leydig cells. Moreover, Sertoli and peritubular myoid cells are crucial for INSL signalling and could underpin further research on the mechanism of cryptorchidism in animal.

Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches.

BACKGROUND: Cryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1-2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1-2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography-tandem mass spectrometry. RESULTS: A PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1-2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age. CONCLUSIONS: The present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

Analysis of transcript and methylation levels of INSL3 and RXFP2 in undescended and descended dog testes suggested promising biomarkers associated with cryptorchidism.

Cryptorchidism is the most common disorder of sex development (DSD) in dogs. This malformation is associated with reduced fertility and with a higher risk of gonadal cancer. Testicular descent is a complex process, and the functions of many environmental and genetic factors are crucial for the proper migration of fetal gonads into the scrotum. Among these, the hormone INSL3 (insulin-like peptide 3) and its receptor RXFP2 (relaxin family peptide receptor 2) play crucial roles in the transabdominal migration of the testes. The genetic background of canine cryptorchidism is poorly elucidated. The aim of this study was to compare the transcript and methylation levels of INSL3 and RXFP2 genes in undescended and descended testes of isolated unilateral cryptorchids, and in gonads of control male dogs with scrotal testes. Next, we searched for polymorphic variants in the 5'-regulatory regions of both genes associated with predispositions to cryptorchidism. The INSL3 transcript level was significantly higher in the undescended testes than in the descended testes of both the affected and control dogs. On the other hand, the mRNA level of RXFP2 was significantly lower in the retained gonads of cryptorchids than in the scrotal testes. The methylation level of a single CpG site located 15 bp upstream of the translation start codon in INSL3 was significantly higher in the testes of the control dogs than in both gonads of cryptorchids. The methylation level of 14 CpG sites in the coding region of INSL3 was significantly higher in undescended testes than in the scrotal testes, which may be associated with the higher mRNA levels of INSL3 observed in these samples. The methylation pattern of two CpG sites in the 5'-flanking region of RXFP2 was similar in both descended and undescended testes. We detected three and seven single nucleotide polymorphisms (SNPs) in the 5'-regulatory regions of INSL3 and RXFP2, respectively. Among these, the frequency of A > C substitution (ss7093349755) located 495 bp upstream of the transcription start site of RXFP2 differed significantly between cryptorchids and control dogs. Our study showed two possible genetic biomarkers associated with canine cryptorchidism: a hypomethylation of a single CpG site in the 5'-flanking region of INSL3, and the ss7093349755 SNP in the 5'-flanking region of RXFP2.

The G178A polymorphic variant of INSL3 may be linked to cryptorchidism among Egyptian pediatric cohort.

Cryptorchidism (CO) is a genital disorder of multifactorial etiology, with serious remote complications. Mutations in insulin-like 3 hormones (INSL3) G/A variant remain a matter of inquiry. We aimed to investigate the association between G178A-INSL3 polymorphism and undescended testis in a cohort of Egyptian children. In this study, a total of 160 children, including 80 cases with primary non-syndromic undescended testes and 80 healthy children with normal external genitalia as controls, both, were analyzed after detailed history, physical examination and imaging for mutations of G178A polymorphism of INSL3 gene by restriction fragment length polymorphism (RFLP) technique. We found most of the undescended testes were inside the inguinal canal mainly on the left side. Genetic analysis revealed that the mutant A allele of G178A INSL3 variant was significantly detected in the patient group with a frequency of 26.2% against 12.5% for control subjects, especially among cases with an evident family history of similar cases as shown by p value = 0.001 and odd's ratio (CI95%) of 0.13 (0.04-0.723). In conclusion, G178A-INSL3 gene polymorphism could be a susceptibility factor for testicular maldescent in Egyptian children. Also, family history of similar cases was considered as significant predictive risk for cryptorchidism, added to the shared genetic links to consanguinity in our locality.

Clinically Differentiated Abnormalities of the Architecture and Expression of Myosin Isoforms of the Human Cremaster Muscle in Cryptorchidism and Retractile Testis.

AIM: To describe architecture and expression of myosin isoforms of the human cremaster muscle (CM) and to individuate changes in clinically differentiated abnormalities of testicular descent: cryptorchidism or undescended testis (UDT) and retractile testis (RT). BACKGROUND: The CM is a nonsomitic striated muscle differentiating from mesenchyme of the gubernaculum testis. Morphofunctional and molecular peculiarities linked to its unique embryological origin are not yet completely defined. Its role in abnormalities of testicular descent is being investigated. SUBJECTS AND METHODS: Biopsy samples were obtained from corrective surgery in cases of cryptorchidism, retractile testis, inguinal hernia, or hydrocele. Muscle specimens were processed for morphology, histochemistry, and immunohistology. RESULTS AND CONCLUSIONS: The CM differs from the skeletal muscles both for morphological and molecular characteristics. The presence of fascicles with different characterization and its myosinic pattern suggested that the CM could be included in the specialized muscle groups, such as the extrinsic ocular muscles (EOMs) and laryngeal and masticatory muscles. The embryological origin from the nonsomitic mesoderm is, also for the CM, the basis of distinct molecular pathways. In UDT, the histological alterations of CM are suggestive of denervation; the genitofemoral nerve and its molecular messengers directed to this muscle are likely defective. Compared with the other samples, RT has a distinct myosinic pattern; therefore, it has been considered a well-defined entity with respect to the other testicular descent abnormalities.