Huifang Kang

Mammalian tissues are characterized by both shared and tissue-associated molecular programs, which together support fundamental biological processes and specialized physiological roles. In reproductive and metabolic tissues, the male reproductive tract and adipose tissues from both sexes exhibit pronounced spatial and functional heterogeneity, while retaining common transcriptomic features across related tissues. Targeted genetic perturbation offers an opportunity to investigate how these shared and tissue-specific molecular programs respond to altered biological conditions. A clearer understanding of baseline transcriptomic organization, together with the consequences of defined gene loss, is therefore essential for elucidating the common and tissue-associated regulatory features that coexist across mammalian systems. In this thesis, transcriptomic profiling of wild-type tissues, with a focus on gene expression and alternative splicing, was combined with CRISPR/ Cas9-based knockout models to investigate transcriptomic organization under physiological conditions and transcriptional responses to genetic perturbation in vivo. This thesis addressed two closely related themes. The first was the characterization of transcriptomic variation across wild-type mouse tissues, including the testis–epididymis axis and major adipose depots in both sexes, with particular emphasis on gene expression and alternative splicing. The second was the investigation of transcriptional responses to targeted CD52 deficiency in knockout mice across the male reproductive tract and the liver in both sexes. CD52 was selected as the target gene because it has been reported in reproductive and immune contexts, and the resulting molecular responses were further examined for shared and tissue-associated response patterns. By integrating descriptive transcriptomic analyses with genetic perturbation studies, this thesis aimed to connect molecular landscape mapping with functional interpretation. Chapter 2 characterized the transcriptomic landscape of the wild-type mouse testis, caput epididymis, corpus epididymis, and cauda epididymis. Comparative analyses revealed clear regional specialization along the male reproductive tract at both the gene expression and alternative splicing levels. The most pronounced molecular transition occurred between the testis and caput epididymis, consistent with the transition from spermatogenesis to post-testicular sperm maturation. Alternative splicing was active in all four tissues, whereas the overlap between differentially expressed genes and differentially spliced genes was limited, indicating that transcriptional and post-transcriptional regulation make partly distinct contributions to tissue specialization. These findings established a transcriptomic framework for understanding spatial regulation and coordinated molecular transitions along the mouse male reproductive tract. Chapter 3 extended transcriptomic profiling to adipose biology through the construction of a multilayer atlas of three major adipose depots, gonadal white adipose tissue (gWAT), inguinal white adipose tissue (iWAT), and interscapular brown adipose tissue (iBAT), in both male and female mice. Integrated analyses of gene expression, alternative splicing, and co-expression patterns showed that transcriptomic differences among adipose depots within the same sex were more pronounced than sex-associated differences within the same depot, with sex-related differences being most evident in gWAT. These findings highlighted distinct multilayer transcriptomic patterns across adipose depots and sexes, thereby extending the thesis beyond the reproductive system toward a broader view of tissue specialization in mammalian biology. Chapters 4 and 5 examined the transcriptomic consequences of CD52 deficiency using CRISPR/Cas9-generated knockout mice. Chapter 4 focused on region-specific transcriptional responses to CD52 loss across the male reproductive tract. CD52-knockout males remained fertile and showed no obvious histological abnormalities; nevertheless, bulk RNA sequencing revealed clear region-resolved transcriptomic alterations across the testis and epididymal segments. The corpus epididymis, in particular, displayed a response pattern distinct from the other tissues, further highlighting regional heterogeneity in the molecular consequences of CD52 loss. Chapter 5 investigated the effects of CD52 deletion in the livers of male and female mice. No visible liver damage or lipid accumulation was observed, transcriptomic profiling revealed a broadly similar hepatic response in both sexes, characterized by consistent changes in genes related to cytoskeletal organization and motor protein function. Together, these chapters showed that deletion of a single gene can induce clear molecular responses that are largely shared across tissues yet retain region-specific features, even in the absence of obvious morphological or reproductive abnormalities. Chapter 6 integrated these findings within a broader biological framework. The General Discussion emphasized that gene expression and alternative splicing represent two regulatory layers contributing to tissue differentiation and functional diversity, and that knockout based transcriptomic analyses can reveal molecular consequences of gene loss beyond overt phenotypic observation. Across the studies presented in this thesis, both baseline molecular organization and responses to genetic perturbation were influenced by tissue identity and physiological context. Overall, this thesis demonstrates the value of combining transcriptomic characterization under physiological conditions with targeted genetic perturbation to help understand tissue specialization and molecular regulation in mammalian systems. By linking descriptive and functional analyses across reproductive, adipose, and hepatic tissues, this work provides a broader framework for examining how tissue-associated molecular programs are organized and how they respond to genetic disruption in vivo. As discussed in Chapter 6, this framework may also support future studies in disease mechanism research and in animal breeding and genomics, including related work in livestock species.