Spermatogonial stem cells (SSCs) constitute a rare population of undifferentiated germ cells responsible for sustaining spermatogenesis throughout life [1]. Beyond their critical role in maintaining spermatogenesis, SSCs are now regarded as valuable tools in reproductive biotechnology, especially in species conservation efforts [2], [3].

While SSC biology has been extensively studied in rodents and primates, its molecular characteristics in felids remain poorly defined [4], [5]. Domestic cats (Felis catus) represent an important translational model [6], [7] for endangered wild felids, many of which are facing severe population declines due to habitat loss, hunting and genetic bottlenecks [4], [8], [9], [10].

During postnatal development, testes undergo significant morphological and functional changes. Prepubertal cat testes lack well-formed lumens and are composed predominantly of immature germ cells such as gonocytes. In contrast, pubertal testis exhibits fully differentiated seminiferous tubules, formation of the blood-testis barrier, and initiation of complete spermatogenesis [11]. While the histological aspects of feline testicular development have been described, molecular markers specific to feline SSCs have not been thoroughly elucidated.

Several surface proteins such as Fibroblast Growth Factor Receptor 3 (FGFR3), Integrin β1 (ITGB1), and Chemokine Receptor Type 4 (CXCR4), have been proposed as SSC markers in other species [12], [13], [14], [15]. FGFR3 is implicated in maintaining stemness and regulating early germ cell development [16]. ITGB1 is known to mediate cell adhesion and interaction with the niche components, facilitating SSC maintenance at the basal membrane of seminiferous tubules [17]. CXCR4, interacting with its ligand SDF-1 (CXCL12), plays a crucial role in germ cell migration, localization, and niche homing [12], [18]. Nevertheless, the expression dynamics of these markers in feline testes during different developmental stages remain largely unexplored (Supplementary table for surface markers).

In functional studies, these pathways are essential for maintaining male fertility. Distribution of the CXCL12-CXCR4 axis leads to failed SSC homing, loss of undifferentiated germ cells and impaired spermatogenic recovery in transplantation models [12], [18], [48], [49], [50]. Loss of ITGB1 (β1-integrin) disrupts spermatogonial adhesion to the basement membrane, compromises Sertoli-germ cell interactions and alters seminiferous epithelial morphology [13], [17], [31]. FGFR3/FGF2 signaling supports SSC self renewal, and its dysregulation has been linked to germline defects and impaired sperm function in human studies [14], [16], [46]. Thus, deficiencies in these pathways result in SSC depletion, abnormal tubule structure or reduced sperm motility [45], underscoring their relevance to fertility. Although SSC markers such as PLZF, UCHL1, DDX4, DBA lectin, SSEA antigens and FoxO1 have been described in domestic cats and wild felids [4], [20], [22], [23], [24], [57], [58], [59], [60], [61], [62], to date there are no published receptor level localisation of FGFR3, ITGB1 or CXCR4 in feline testis. Existing literature show clear roles for FGFR3 in undifferentiated spermatogonia and germline homeostasis [14], [16], ITGB1 , in SSC niche adhesion and basal membrane anchoring [13], [17], and CXCR4 in SSC homing, migration and maintenance of the undifferentiated pool [12], [18], [48], [49], [50]. However, their developmental expression patterns in felids remain unknown.

Therefore, the present study aimed to examine the expression morphology and immunolocalisation of FGFR3, ITGB1, and CXCR4 in prepubertal and pubertal domestic cat testes. Immunofluorescence analysis combined with quantitative image-based evaluation was employed to determine the developmental dynamics of these markers and to assess their potential utility in identifying feline SSCs. This approach enabled the identification of the stage-specific expression profiles and highlighted their potential as SSC markers in cats. The findings establish a molecular foundation that may support the development of SSC-based reproductive technologies and contribute to conservation efforts in endangered felid species.