Exploring Testicular Descent: Recent Findings and Future Prospects in Canine Cryptorchidism

Cryptorchidism is a congenital disorder of sex development manifested by the failure of testicular descent. The form of cryptorchidism is classified based on the location of the testes (abdominal or inguinal) and the number of affected gonads (bilateral or unilateral). Unilateral cryptorchidism can be further categorized as right- or left-sided. In both humans and dogs, cryptorchidism leads to subfertility or infertility [1] and an increased risk of testicular malignancy [2, 3]. In dogs, the risk of malignancy in undescended testes is up to 13.6-fold higher [4], with the most common cancers being seminomas, interstitial cell tumors, and Sertoli cell tumors [5]. Right-sided inguinal cryptorchidism is the most common form [6], particularly in small breed dogs, while abdominal cryptorchidism is often diagnosed in medium and large breed dogs [7]. It is worth noting that the higher incidence of right-sided cryptorchidism may be due to anatomical features, as the right gonads develop in a more cranial position than the left gonads. Consequently, they have to cover a longer distance from their primary location to the internal ring of the inguinal canal and then to the scrotum [7].

In humans, isolated cryptorchidism is diagnosed in 5% of newborn boys, but the testes often descend spontaneously into the scrotum within the first few months of life, reducing the prevalence to approximately 1% [8]. In dogs, cryptorchidism is the most common form of disorder of sex development, with an incidence ranging from 0.8% to 9.7%, depending on the breed [6]. Both the breed-dependence and high prevalence of this condition are linked to the intensive breeding of purebred dogs. Selection leads to a systematic reduction of heterozygosity in a population [9], increasing the incidence of both desirable and deleterious recessive traits. For this reason, purebred dogs serve as important models for studying genetic diseases [10].

Undescended testes are a common abnormality in various syndromes, including persistent Müllerian duct syndrome, androgen insensitivity syndrome, and other conditions such as Arboleda-Tham syndrome [11‒13]. In addition, abnormal development of the epididymo-testicular junction results in abdominal cryptorchidism in humans [14, 15]. Isolated cryptorchidism is considered a defect with a complex etiology. In dogs, the heritability of cryptorchidism was found to range from 0.11 to 0.23, depending on the method of analysis [16], and was 0.26 in pigs [17]. These values indicate that while genetic factors play an important role in the etiology of cryptorchidism, other factors – such as environmental, maternal, and epigenetic – are also involved [18]. Nevertheless, the search for DNA variants associated with cryptorchidism in dogs is warranted in order to limit the spread of this defect.

The purpose of this review was to summarize and discuss recent findings related to canine cryptorchidism. Based on these findings, a new line of research is proposed to study the development of the epididymo-testicular units and their junction in dogs affected by cryptorchidism.

Bipotential gonads develop as urogenital ridges from the coelomic epithelium on the ventral surface of the mesonephroses. In addition to these primordial gonads, Wolffian ducts (WDs) and Müllerian ducts (MDs) also form in both XY and XX fetuses. In XY mammalian fetuses, expression of the SRY gene induces testicular development. The WDs give rise to the epididymis, vas deferens, and seminal vesicle, while the mesonephroses regress and their remnants contribute to the formation of the rete testis (RT) and efferent ducts (EDs) [19]. The RT is a network of tubules within the testis that serves as an important conduit connecting the seminiferous tubules to the EDs. The connection between the RT and the EDs has been described in detail [20]. The RT-ED junction plays a crucial role in male fertility by enabling the transport of spermatozoa to the epididymis, where they undergo maturation [21]. In male fetuses, Leydig cells synthesize androgens, which induce the stabilization and differentiation of the WDs. Conversely, the MDs regress upon stimulation with anti-Müllerian hormone (AMH) secreted by Sertoli cells [22, 23]. However, the differentiation of the WDs into functionally distinct regions depends on the expression of the Hoxa and Hoxd (9–13) genes. In particular, the Hoxa10 and Hoxa11 genes play a role in the development of the bounder region between the epididymis and vas deferens, while Hoxa13 and Hoxd13 are involved in seminal vesicle formation in mice [24, 25].

The development of the gonads occurs within the abdominal cavity and the fully developed testes migrate from their original location into the developing scrotum. Testicular descent unfolds in two primary phases: transabdominal and inguinoscrotal [26]. In humans, the transabdominal phase occurs between 8 and 15 weeks of gestation [26, 27], leading to testicular migration toward the inner ring of the inguinal canal. In dogs, it begins around day 53 of gestation [28]. In mice, it occurs between 15.5 and 17.5 embryonic days [27]. The second phase should be completed between 25 and 35 weeks of fetal life in humans [29], by the sixth month of postnatal life in dogs [28], and by the second week of postnatal life in mice [30].

The transabdominal phase of testicular descent depends on the development of the gubernaculum [31, 32]. The gubernaculum is a structure attached to the testis and epididymis and extends to the abdominal wall, as observed in human fetuses [33]. In addition, the gonads are connected to the cranial suspensory ligaments, which regress in males upon androgen stimulation [26]. Development of the gubernaculum depends on insulin-like 3 hormone (INSL3), secreted by fetal Leydig cells, which acts via the RXFP2 receptors present in the gubernaculum [29]. The INSL3-RXFP2 interaction induces dynamic changes in the gubernaculum [26, 27, 34], including its elongation and the formation of a “gubernacular bulb” at the caudal end. Morphological and developmental differences between humans and dogs compared to rodents have been described [26, 34]. In humans and dogs, the connective tissue of the gubernaculum undergoes remodeling, eventually becoming a fibrous structure containing collagen fibers, as noted by Arrighi et al. (2010). Due to the lack of muscle, this structure is probably not capable of active contraction in these two species. Furthermore, cell proliferation and an increase in the extracellular matrix – mainly consisting of hydrophilic molecules – lead to the swelling reaction and formation of the gubernacular bulb in humans and dogs [26]. Their gubernaculum is described as a jelly-like/gelatinous structure [33, 34]. On the other hand, the rat gubernaculum consists of a muscular outer layer with a mesenchymal core. The caudal part of the rat gubernaculum undergoes intense proliferation, and these cells differentiate into new cremaster muscle cells [26]. During testicular descent, rhythmic contractions have been found in rats after stimulation with calcitonin gene-related peptide (CGRP) probably due to the presence of smooth muscle [35]. A major role of Insl3 in this process has been confirmed in studies using knockout mice, in which abdominal gonads were observed [36]. Moreover, overexpression of Insl3 in transgenic female mice induced ovarian migration toward the abdominal wall [37].

The second phase of testicular descent is thought to depend on androgen action via androgen receptors (ARs) [26]. Supplementation of pregnant female mice with flutamide, an AR antagonist, disrupted the growth of the gubernaculum [38]. Additionally, cases of androgen insensitivity syndrome often exhibit inguinal cryptorchidism [12]. In rodents, testosterone indirectly influences the second phase by affecting the genitofemoral nerve, which subsequently secretes CGRP. Impaired CGRP secretion leads to cryptorchidism in rodents [39], although this mechanism has not been confirmed in humans [29]. The role of estrogens in abnormal gubernacular development and impaired testicular descent has also been investigated [40]. Specifically, studies have examined the effect of endocrine disrupting chemicals on androgen-estrogen imbalance in males. Research has shown an association between fetal exposure to estrogens, such as diethylstilbestrol, and an increased risk of human cryptorchidism [41].

Human isolated cryptorchidism has been associated with DNA variants in several genes: INSL3 [42, 43], RXFP2 [42, 44], AR [42], HOXA10 [45], HOXD13 [46], and AXIN1 [47]. In addition to searching for causative mutations, mRNA and protein levels have also been analyzed. Reduced expression levels of the FGFR1, SOS1, and RAF1 genes were found in human cryptorchid testes [48]. Moreover, researchers focused not only to gene expression in human cryptorchid testes but also in structures such as the appendix testis (AT) and the gubernaculum (G). The AT, a vestigial structure derived from the MDs, has also been investigated in relation to cryptorchidism. Studies found no association between the length of this structure and an increased risk of cryptorchidism [49], or with serum AMH levels [50]. However, a positive correlation was observed between the length of the AT and serum INSL3 levels [51], although this was not confirmed in another study [50].

Since reproductive organ development and testicular descent occur during fetal life, analyzing hormone concentrations or gene expression levels in non-rodent fetuses is challenging. Therefore, amniotic fluid samples collected from healthy and cryptorchid live-born boys during routine amniocentesis were used [52]. At 13–16 weeks of gestation, INSL3 levels in amniotic fluid were notably higher in cryptorchid cases compared to controls. A nonsignificant decrease in INSL3 levels was observed between 17 and 22 weeks in cryptorchids, with a similar pattern found in cases of hypospadias.

Another study on INSL3 mRNA expression in fetal testes showed that INSL3 overexpression begins as early as 7–8 weeks of gestation [53]. A recent study found that INSL3 levels in umbilical cord blood collected between 15 and 20 weeks of gestation were 5 to 100 times higher than those detected in amniotic fluid samples. This report also indicated that INSL3 mRNA levels were minimal in fetal testes at 14–16 weeks, peaked at 18 weeks, and then decreased before increasing again in adult testes [54]. Notably, in mice with a heterozygous targeted disruption of the Insl3 gene, testicular descent occurred but was delayed [55]. These findings revealed the importance of the timing and levels of INSL3 expression during testicular descent.

The findings suggest that male fetuses destined to develop cryptorchidism initiate INSL3 synthesis earlier in gestation, but that protein concentrations decrease as gestation progresses. Studies also indicate that INSL3 expression is highly regulated, similar to other key factors involved in male sex determination and development (e.g., SRY, SOX9, FGF9, AMH). High levels of INSL3 synthesis must occur within the specific time window corresponding to the first phase of testicular descent.

A recent review on canine cryptorchidism provided an update on our understanding of both the clinical aspect and the potential molecular events associated with this condition [56]. Since then, numerous new studies have been performed (summarized in Table 1). The present review briefly describes the results of those studies and delves into a more comprehensive discussion to provide prospective directions for further research.

Dogs with cryptorchid testes showed significantly higher AMH levels in serum and local spermatic venous blood samples [57, 58]. AMH levels reflect the fetal Sertoli cell function and are negatively regulated by testosterone [69]. Notably, AMH levels are used as a marker of bilateral cryptorchidism in boys, with high levels being associated with this condition [70].

In contrast, INSL3 secretion by Leydig cells occurs in adult males, and the amount of INSL3 reflects the number and functionality of Leydig cells [71]. Cryptorchid cases representing different species showed reduced serum INSL3 levels [59]. In addition, INSL3 levels were lower in cord blood samples collected from new born cryptorchid boys [72]. In dogs, cryptorchid testes were smaller and produced lower amounts of INSL3 peptide (per testis) compared to scrotal gonads. However, INSL3 levels (per mg protein) were increased in retained testes and were consistent with relative mRNA levels [63].

Based on these findings and the study by Anand-Ivell et al. (2018), it appears that INSL3 secretion is prematurely elevated during fetal development and then decreases below normal levels in males destined to develop cryptorchidism. INSL3 secretion is likely to be reduced in prepubertal and pubertal males due to a lower number of adult Leydig cells in retained, underdeveloped testes.

The first study on testosterone concentrations in cryptorchid dogs (unilateral inguinal and unilateral abdominal cases) showed no differences in either the peripheral or spermatic venous blood samples compared to the controls [73]. In a recent report, unilateral cryptorchidism in dogs was associated with lower testosterone levels in both the peripheral and spermatic venous blood samples [58].

The neuropeptide orexin A (OXA) and its receptor (OX1R) are involved in various biological processes, including lipid metabolism, food intake, and sleep, and are present in canine testes [74]. In vitro analysis of fresh testis slices showed that OXA-OX1R interaction stimulates steroidogenesis in both scrotal and undescended gonads, due to their expression in Leydig cells. This effect of OXA-OX1R signaling on steroidogenesis was further confirmed [75]. In undescended gonads, signals for OXA were observed in Sertoli cells and gonocytes, while OX1R was found in Sertoli cells but absent in spermatocytes and spermatids [74]. Another study examined the role of orexin B (OXB) and its receptor (OX2R) [76]. It was found that OXB-OX2R signaling does not stimulate testicular steroidogenesis. Additionally, undescended gonads lacked OXB and OX2R expression in spermatocytes and spermatids. The presence of these receptors in the scrotal gonads suggests that OXA-OX1R and OXB-OX2R signaling may play a role in spermatogenesis. However, these studies do not contribute to our understanding of the genetic causes of cryptorchidism in dogs.

A comprehensive study of testicular and epididymal samples from adult dogs, including 10 unilateral cryptorchids and 10 controls, was performed for these three estrogen receptors [77]. Canine cryptorchidism was associated with increased expression of GPER and ESR1 in the testicular-epididymis unit, while ESR2 expression was reduced in the epididymis. ESR1 is crucial for fluid reabsorption and sperm maturation in the epididymis [78], while Esr2 is involved in germ cell cycle arrest and apoptosis before puberty in mice [79, 80]. Higher ESR2 expression has been linked to reduced sperm quality and infertility in boars [81]. In rat Sertoli cells, estrogens act through ESR2 to inhibit proliferation and stimulate Sertoli cell maturation [82]. Therefore, changes in the expression of estrogen receptors likely result from disruptions in spermatozoa development and maturation in the undescended testes. Although these findings may contribute to our understanding of spermatogenesis, they do not shed light on the cause of cryptorchidism.

Testosterone plays a crucial role in testicular descent and impaired steroidogenesis is one of the deleterious effects of cryptorchidism. Therefore, mRNA expression and DNA methylation levels for these steroidogenic genes were analyzed in scrotal (C) testes from control dogs, as well as both gonads (D-scrotal and UD-inguinal) from cryptorchid dogs [60]. Undescended gonads showed a significant increase in CYP17A1 mRNA expression, while CYP19A1 mRNA levels were reduced compared to both scrotal testes (C and D). In addition, the DNA methylation level of a single CpG site within the promoter region of CYP17A1 was decreased in undescended testes and negatively correlated with mRNA expression. These changes are likely due to the abnormal environment in the inguinal canal. This study was the first to demonstrate an epigenetic modification associated with cryptorchidism in dogs.

Some genes involved in temperature-dependent sex determination, such as cyp19a in the European sea bass [83], are regulated by changes in DNA methylation levels. A study in mice showed that hepatic Cyp17a1 is expressed in a sex-dependent pattern and is regulated by sex-dependent methylation [84]. Variable DNA methylation patterns are directly influenced by transcriptional regulators such as DNA methyltransferases (DNMT3A and DNMT3B). A recent RNA-sequencing (RNA-seq) study in canine testes showed that the inguinal location of gonads was associated with significantly reduced mRNA expression of DNMT3B and DNMT1, whereas DNMT3A expression was markedly increased [61]. The promoter region of CYP17A1 is regulated by DNMT3a-dependent methylation, while SP1 overexpression inhibits the binding of this methyltransferase, increasing CYP17A1 expression in glioma [85]. However, cryptorchidism had no effect on SP1 expression in dogs. In contrast, another well-known regulator of CYP17A1 expression, steroidogenic factor 1 (SF-1) encoded by NR5A1, was significantly increased in inguinal gonads of dogs, similar to the CYP17A1 gene [60, 61].

The CYP17A1 gene encodes an enzyme involved in several steps of steroidogenesis. The CYP17A1 protein catalyzes two reactions in the conversion of pregnenolone to dehydroepiandrosterone, an androgen precursor [86]. The negative correlation between mRNA and DNA methylation of the CYP17A1 gene in the inguinal gonads suggests that epigenetic changes may be involved in a compensatory mechanism to increase CYP17A1 expression and promote testicular steroidogenesis.

The relative mRNA expression levels of estrogen receptor 1 (ESR1) and AR were examined in the same material as described by Krzeminska et al. [62] (2020) and reported in a PhD thesis, then confirmed by RNA-seq analysis [61]. ESR1 expression was low in all gonads (UD, D and C). It should be noted that the mRNA analyses were based on material containing all cell types: Leydig cells, Sertoli cells, and others. The results are therefore not consistent with the immunostaining analyses [77]. In contrast, mRNA levels of the AR gene were significantly elevated in the inguinal gonads, but DNA methylation levels of the promoter region of the AR were low and similar in all gonads. The elevated AR mRNA levels in the inguinal gonads were probably associated with Sertoli cell hyperplasia, a phenomenon previously described in the undescended testes of dogs [87]. However, further expression analyses should be carried out on isolated cells or using spatial transcriptomics.

The mRNA expression levels of these two key genes for testicular descent were examined in the inguinal (UD) testes, as well as the scrotal testes of cryptorchid (D) and control (C) dogs [64]. INSL3 levels were significantly increased in the undescended gonads compared to the scrotal testes (D and C), similar to the report by Hannan et al. (2015). Previously, however, total INSL3 mRNA levels were reduced in undescended testes (calculated per testis). In turn, RXFP2 mRNA levels were significantly reduced in UD testes. DNA methylation levels for these genes were also examined. A single CpG site in the 5′UTR region of INSL3 was significantly hypermethylated in testes of the controls compared to the inguinal and scrotal testes of cryptorchid dogs. DNA methylation of the 5′ flanking region of RXFP2 was hypermethylated and similar in UD, D, and C testes [64].

The promoter region of canine INSL3 was hypomethylated in both gonads of unilateral cryptorchid dogs. Therefore, it was considered the first epigenetic marker for canine cryptorchidism found in gonads and not in blood samples [68]. The lack of a similar DNA methylation pattern in blood samples indicates that this epigenetic event is testis-specific. However, mRNA levels were only increased in the inguinal gonads. Several questions therefore arise:

• (1)

  Is it possible that adult Leydig cells reflect the methylation pattern of fetal Leydig cells? (See review on three models of Leydig cell origin and development [88]).

• (2)

  Could hypomethylation be associated with an increase in INSL3 expression during fetal life? (The highest expression not at the optimal time).

• (3)

  Is it possible that the right and left fetal gonads show a similar methylation pattern and thus expression profile, but only the right gonad does not pass through the inguinal canal? (Due to its greater distance from the original position to the scrotum).

• (4)

  Is it possible that the higher mRNA levels are due to a compensatory mechanism to increase INSL3 secretion in the undescended testes? Nevertheless, the undescended gonads do not produce similar amounts of INSL3 protein compared to the scrotal testes due to smaller size/fewer Leydig cells.

• (5)

  Why could hypomethylation increase mRNA levels in the undescended gonads, but not in scrotal testes from cryptorchids? What other mechanism is used by Leydig cells of the undescended gonads to increase INSL3 expression?

Given these concerns, greater emphasis should be placed on understanding the regulation of INSL3 expression, propeptide processing, and peptide secretion by both fetal and adult Leydig cells. The study by Nowacka-Woszuk et al. (2020) suggests a potential role for epigenetic modifications in testicular descent, highlighting the need to investigate INSL3 regulation. Advanced techniques, such as spatial transcriptomics and genome-wide DNA methylation arrays, should be applied to fetal Leydig cells to gain a deeper understanding of INSL3 secretion during testicular descent. It is important to note that studies on adult Leydig cells may not provide reliable insights into the mechanism of INSL3 expression during testicular descent.

The transcriptome sequencing method applied for the inguinal (UD) and scrotal testes from both cryptorchid (D) and control dogs (C) showed global changes in mRNA expression. Specifically, 8,028 and 7,619 differentially expressed genes were identified for UD versus D and UD versus C comparisons, respectively [61]. However, no differentially expressed genes were observed between D and C gonads. The expression levels of eleven genes were verified using qPCR and summarized in Table 1. Moreover, DNA methylation levels were analyzed for these selected genes. Overall, the expression of the TP53 and KITLG genes was increased in inguinal gonads. Conversely, a decrease in KLF4 mRNA levels was observed, correlating with increased methylation levels in the promoter region. The role of TP53 in cancer development is well understood [89]. The KITLG gene controls gametogenesis, including primordial germ cell development, and is associated with a higher risk of testicular germ cell tumors [90]. KLF4 acts as a suppressor of colorectal cancer cell proliferation [91], while in breast tumors, miRNA-dependent downregulation of KLF4 is associated with inhibition of tumor development [92]. These changes in expression levels may reflect the tumorigenesis in undescended gonads. In addition, genes involved in spermatogenesis were significantly downregulated in the undescended gonads, whereas AMH mRNA was increased, which is consistent with both serum and testicular AMH levels in cryptorchid dogs [57, 58]. Moreover, the expression of the KAT6A gene, the role of which is elucidated below, was decreased specifically in the inguinal gonads [61].

A search for a causative mutation for canine cryptorchidism was carried out for more than 100 dogs with cryptorchidism. Among these, there were 40 cases with abdominal cryptorchidism. Unfortunately, Sanger sequencing of the coding sequence and the promoter region did not reveal any potential causative DNA variant in cryptorchid dogs [64, 65]. Although the INSL3 gene is one of the most important candidate genes for cryptorchidism, its mutations are identified in only 1–2% of humans with cryptorchidism [93]. It appears that if the INSL3 gene is involved in the etiology of cryptorchidism, the effect is more likely related to its expression level than to DNA polymorphism.

The RXFP2 gene consists of 18 exons, so a preliminary DNA sequencing of the coding region was performed using mRNA isolated from the testes, specifically from abdominal gonads located near the kidneys. No deleterious DNA variant was found in cryptorchid dogs (unpublished data). However, a DNA variant in the 5′flanking region was identified, and it was significantly more frequent in cryptorchid dogs. In silico analysis indicated that this variant could potentially affect the binding of testicular transcriptional factors (HMGA1, EP300, STAT4, STAT6, and ELK1) [64].

Given the significant divergence in mRNA expression between the inguinal and scrotal testes for the CYP17A1 and CYP19A1 genes, Sanger resequencing of their 5′flanking regions was performed. Two DNA variants were identified: a SNP polymorphism (g.15299258C>T, rs23416465) in CYP17A1 and an insertion/deletion polymorphism (g.16988911_16988912insA, rs850623615) in CYP19A1. Neither variant showed an association with an increased risk of canine cryptorchidism [60].

To search for DNA polymorphisms, both the 5′flanking region and the coding sequence of canine ESR1 were resequenced, initially for 33 cryptorchid and 35 healthy dogs. Altogether, seven DNA variants were identified. The SNP variant in exon 5 (g.42208686A>G; p.Ile327Val) was then selected to evaluate the distribution of two alleles (A and G) in larger cohorts of dogs (91 cryptorchids and 108 controls). Odds ratio testes revealed no significant differences in the frequency of these two alleles in the two included groups [62].

There are tandemly repeated trinucleotide motifs (CAG and GGC) in exon 1 of the AR gene, and the number of repeats is variable. Since the second phase of testicular descent is controlled by testosterone, the association of this polymorphism with cryptorchidism was analyzed. A higher number of repeats has been associated with the risk of non-syndromic (isolated) cryptorchidism in humans [94, 95]. However, this association was not confirmed in dogs with isolated cryptorchidism [65] or cryptorchidism coexisting with hypospadias [66].

A genome-wide association study (GWAS) was performed in cryptorchid Siberian Husky dogs [96] and discussed in a previous report [56]. A recent GWAS analysis was applied to large cohorts (3,736 cryptorchid dogs and 3,736 control dogs) representing several breeds [67]. The results showed that a region of chromosome 10 (CFA10) is associated with the risk of inguinal cryptorchidism in dogs. Further analyses identified a SNP polymorphism in the 5′UTR of the HMGA2 gene. This gene is responsible for body weight variance in dogs [97]. Moreover, Hmga2 knockout mice showed a pygmy/dwarf phenotype [98]. However, no association of this variant with body weight or breed of cryptorchid dogs was found.

Given the higher prevalence of inguinal cryptorchidism in small breed dogs, the association of the HMGA2 gene with the risk of inguinal cryptorchidism in dogs appears to be an important result. The authors performed analyses based on the body weight of dogs (previously HMGA2 was linked to body weight). However, body weight is influenced by the size of the dog and the degree of its muscularity. Does the drastic reduction in size of some breeds and all their organs (compare Chihuahua and the wolf as an ancestor) lead to an impaired migration of the testis through the inguinal canal? On the other hand, is it possible that the higher incidence of abdominal cryptorchidism in large breed dogs is due to:

• (1)

  A greater distance from the original position of the testis to the inner ring of the inguinal canal and insufficient gubernacular growth?

• (2)

  The growth of the gubernaculum or the development of the connection of the epididymo-testicular unit with the gubernaculum being insufficient to pull down the large testis?

The results of RNA-seq analysis on canine testes were used to search for DNA variants in mRNAs [61]. Six missense DNA variants, out of 20,366 total SNPs, showed significant differences between cryptorchids and controls. A DNA variant in exon 17 of the KAT6A gene (c.3689C>T, p.Ala1230Val) and a co-segregated SNP in the 3′UTR region (16:23716292G>A) were resequenced in larger cohorts and showed an association with canine cryptorchidism. In silico analysis revealed neither a deleterious effect of the SNP in exon 17 nor any role of the SNP in the 3′UTR region miRNA binding. However, the missense SNP was located within the unstructured region of KAT6A, which is crucial for its enzymatic activity. The KAT6A gene encodes a histone acetylase that is part of a protein complex involved in the epigenetic control of developmental processes, including HOX gene expression. Since the KAT6A governs the acetylation of lysine 9 of histone H3 (H3K9), the researchers set out to validate the enzymatic activity of both wild-type and mutant proteins in vitro. This yielded a valuable result, providing significant progress in our understanding of canine cryptorchidism. TT/AA genotypes (exon 17/3′UTR) were found to be associated with reduced H3K9 acetylation in all gonads (UD, D, and C). This association between genotype and acetylation levels was found in both gonads from cryptorchid. However, it was particularly pronounced in the undescended gonads, probably due to the general abnormal function of the inguinal gonads.

The SNP in exon 17 of the KAT6A gene, together with a decrease in acetylation activity, paved the way for a new direction in studies on canine cryptorchidism. Stachowiak et al. (2024) discussed a potential link between the KAT6A gene and cryptorchidism. They referred to Arboleda-Tham syndrome in humans caused by mutations in the KAT6A gene. Some patients with this syndrome manifested cryptorchidism as a coexisting symptom [13]. They mentioned that KAT6A controls HOX genes expression [99, 100] and referred to the role of both Hoxa10 and Hoxa11 in the development of cryptorchidism in knockout mice [101]. It is worth stressing here that DNA variants in HOXA10 [45] and HOXD13 [46] have been associated with a higher risk of cryptorchidism in humans. The link between the HOX genes and epididymal development was highlighted in the section discussing “testicular development and descent.” Furthermore, comprehensive reports on epididymo-testicular descent have been previously published [102, 103]. They showed that testicular descent is preceded by descent of the body and tail of the epididymis. Abnormal development of WDs, including epididymal disjunction, can mainly lead to abdominal cryptorchidism in humans [14, 15, 104, 105]. In mice, the absence of the epididymis precludes testicular descent [106].

Since the gubernaculum is connected to the epididymis and testis, and the KAT6A gene is involved in epididymal development, the search for molecular causes of canine cryptorchidism should be directed toward studying the development of epididymis and the connection between the epididymis and the testis.

A vast majority of papers have included analyses of the testes (and sometimes the epididymis), but little attention has been paid to the development of the epididymis itself and its connection to the testis and the gubernaculum. It is therefore recommended that comprehensive studies of the epididymis and the epididymo-testicular unit (ET) in cryptorchid and control dogs be performed, including histological, epigenetic, and genetic studies such as spatial transcriptomics and DNA sequencing.

The association between global H3K9 acetylation and KAT6A polymorphism suggests that a much deeper analysis of epigenetic modifications is warranted to enhance our understanding of the pathogenesis of canine cryptorchidism. In addition, the link between the HMGA2 gene and inguinal cryptorchidism in dogs indicates that intensive selection in dog breeding is a concerning phenomenon. The size of the dog, along with drastic changes in the morphology of small and large breeds, appears to interfere with a complex process of testicular descent. Studies in human amniotic fluid have advanced our knowledge of INSL3 expression during the initiation of the transabdominal phase of testicular migration. High levels of INSL3 expression at the optimal time window are necessary for successful descent of the testes. The promoter region of canine INSL3 was hypomethylated in both gonads of unilateral cryptorchid dogs. The regulation of canine INSL3 expression by both fetal and adult Leydig cells should also be considered in further studies.

Overall, whole-genome sequencing or a genome-wide association study (GWAS) are powerful tools for identifying disease-associated DNA variants. However, both methods require more complex bioinformatic analysis and larger cohorts of cases and controls to produce reliable results. On the other hand, whole-exome sequencing targets DNA variants within all coding sequences but excludes regulatory regions. RNA sequencing focuses exclusively on sequences of transcribed genes.

Recent studies have extensively applied expression analysis in the undescended gonads of adult dogs. RNA-sequencing results have confirmed that the changes in mRNA expression are a consequence of the abnormal position of the gonads (as shown in the comparison of descended and control testes). Moreover, these expression analyses, followed by further molecular investigations, led to the identification of a DNA variant (RXFP2) and a DNA methylation pattern (INSL3) associated with canine cryptorchidism.

The RNA-seq has proven to be a valuable tool for studying disease by detecting changes in gene expression and identifying DNA variants in coding sequences. Expression analyses have provided insights into molecular events such as tumorigenesis and defects in spermatogenesis in the undescended gonads. These efforts have also led to the identification of a DNA variant in the coding sequence of a less obvious gene. The KAT6A gene may open a new avenue of research into canine cryptorchidism.

The author has no conflicts of interest to declare.

This study was not supported by any sponsor or funder.

P.K.: writing and editing the manuscript.