Zuyong He

Fertilization and early-cleavage failure are the major causes of assisted reproductive technology failure, but genotype-guided care is underused. We conducted whole-exome sequencing with Sanger confirmation in 20 men from couples with unexplained fertilization and cleavage failure after in vitro fertilization or intracytoplasmic sperm injection (ICSI). Then, further sperm transmission electron microscopy, immunofluorescence, and, where appropriate, embryo quantitative parental contamination test (qPCT) and kinship analysis were integrated in selected cases. Six novel variants were found in four families: two each in dynein axonemal heavy chain 10 ( DNAH10 ) and dynein axonemal heavy chain 12 ( DNAH12 ) and one in cilia and flagella associated protein 47 ( CFAP47 ) and phospholipase C zeta 1 ( PLCZ1 ), respectively. DNAH10/DNAH12 variants segregated with multiple morphological abnormalities of the sperm flagella and showed “9+2” axonemal disruption and reduced dynein signals. Hemizygous CFAP47 c.8006C>T is associated with cleavage failure and altered PLCZ1 localization. Homozygous PLCZ1 c.589C>T in a consanguineous pedigree coincided with multinuclear (≥3 pronuclei) zygotes, and qPCT/kinship indicated excess paternal contribution, consistent with suspected polyspermy in multinuclear zygotes. Management-matched genotype: ICSI restored fertilization in DNAH10/DNAH12 deficiency, whereas ICSI plus assisted oocyte activation (AOA) improved cleavage and yielded ongoing pregnancies in CFAP47/PLCZ1 deficiency. These findings outline potentially actionable clinical strategies–ICSI for axonemal dynein defects and ICSI plus AOA for activation pathway defects–and support targeted genetic testing plus embryo qPCT/kinship in selected cases.

Context: Growth differentiation factor (GDF) 9 is a member of the transforming growth factor (TGF)-β superfamily, which plays an important role in mammalian ovarian follicular development and female fertility. However, its regulatory role in the male reproductive system remains largely unknown. Aims: This study aimed to investigate the regulatory mechanism of GDF9 in porcine testicular development. Methods: We established a GDF9-edited pig model through CRISPR-Cas9 technology and somatic cell nuclear transplantation. The testicular development of one edited piglet at birth and one at Day 14 were characterized via Western blotting, haematoxylin–eosin staining, and immunohistochemistry at cellular and molecular levels. Key results: Based on the preliminary analysis of one edited piglet at birth and one at Day 14, we did not observe changes to the gross morphological and anatomical structures of porcine testes at early developmental stages. However, the developmental process or cellular functions of Sertoli cells, Leydig cells and spermatogonia may be affected, as reflected by the altered expression patterns of SOX9, AR, DHRS9, DDX4 and ANXA2 in the testes. Noticeably, the altered expression of StAR may indicate an impact of GDF9 editing on the steroid hormone synthesis functions of porcine Leydig cells. Conclusions: These findings indicate that GDF9 may affect porcine testicular development at the early postnatal stage. Implications: These preliminary results provide a valuable foundation for future comprehensive studies toelucidate the regulatory mechanism of GDF9 in porcine testicular development.

Kit encodes a receptor tyrosine kinase crucial for various biological processes. To investigate how Kit structural mutations associated with the porcine dominant white phenotype affect hematopoiesis, we utilized three distinct gene‐edited mouse models: Kit coding sequence (CDS) duplication ( Kit ^{dup /+} ), Kit exon 17 deletion ( Kit ^{D17 /+} ), and a compound heterozygous model carrying both mutations ( Kit ^dup/D17 ), along with wild‐type controls ( Kit ^+/+ ). We observed that the Kit structural mutations significantly impaired erythropoiesis in bone marrow, resulting in hypoplastic macrocytic anemia and compensatory erythropoiesis in the spleen. Transcriptomic analyses revealed that these structural Kit mutations attenuate PI3K and MAPK signaling activation and downregulate genes essential for erythroid differentiation. Our findings provide novel mechanistic insights into how Kit mutations contribute to hematopoietic dysfunction.

Background: A splice mutation that causes skipping of exon 17 in the KIT gene is a major reason for the dominant white phenotype of pigs. Exon 17 of the KIT gene may be related to differences in testis size and sperm quality among different pig breeds. Investigating the effects of exon 17 of the KIT gene on spermatogonia differentiation and testicular development is essential for understanding the genetic causes of reduced fertility and semen quality in pigs. To better understand the effects of the splice mutation of KIT on porcine spermatogenesis, we described an exon 17 deletion mouse model (Kit ^D17/+) constructed by simulating splice mutations in KIT for functional verification. Results: Deletion of exon 17 of Kit severely impaired the differentiation of spermatogonia and promoted the apoptosis of germ cells, resulting in testicular dysplasia and decreased sperm quality and male fertility. Further transcriptomic analysis revealed inhibited expression of genes involved in meiosis and spermatogenesis and attenuated MAPK-ERK signaling in the testicular tissues of Kit ^D17/+ mice. The attenuated MAPK-ERK signaling caused by impaired Kit phosphorylation was confirmed by western blotting. Conclusions: Our study demonstrated that deletion of exon 17 of Kit severely impaired spermatogenesis and testicular development, leading to decreased semen quality and male fertility. These findings verified the function of exon 17 in the Kit gene and provide a theoretical basis for improving the semen quality of dominant white pigs through correction of the splice mutation of KIT.

Whole‐exome sequencing (WES) is frequently utilized in diagnosing reproductive genetic disorders to identify various genetic variants. Canonical ±1,2 splice sites are typically considered highly pathogenic, while variants at the 5′ or 3′ ends of exon boundaries are often considered synonymous or missense variants, with their potential impact on abnormal gene splicing frequently overlooked. In this study, we identified five variants located at the last two bases of the exons and two canonical splicing variants in five distinct families affected by reproductive genetic disorders through WES. Minigene analysis, RT‐PCR and Quantitative Real‐time PCR (RT‐qPCR) confirmed that all seven variants induced aberrant splicing, with six variants altering gene transcriptional expression levels. These findings underscore the crucial role of splice variants, particularly non‐canonical splice sites variants, in reproductive genetic disorders, with all identified variants classified as pathogenic.