Transgenic pig over-expressing leptin as a model for female infertility

Infertility is a growing global health concern affecting millions of couples worldwide. Both overweight and underweight individuals face reproductive issues, resulting in infertility. Due to small size and short life, mice are not necessarily suitable animal models for human infertility. While, pigs having large size, anatomical and physiological similarities with human are considered as more ideal animal model of human disease. Leptin is a well-known adipokine that serves as an endocrine signal between adiposity and fertility. However, the exact mechanisms underlying leptin's effects on reproductive function remain unclear. To shed light on this, our study focused on pigs genetically engineered to overexpress leptin. These leptin-overexpressing pigs exhibited several reproductive abnormalities, including reduced body weight and size, decreased back fat thickness, and displayed late onset of puberty, and irregular estrous behavior characterized by increased inter-estrous interval and more breeding attempts until pregnancy. This reproductive impairment in leptin pigs was followed by hormonal imbalances and altered steroidogenesis. Bulk RNA sequencing of the ovaries revealed neutrophilic infiltration followed by upregulation of inflammation related genes. To gain a deeper understanding of the cellular levels, we employed single-nucleus RNA sequencing (snRNA-seq), and found that leptin overexpression triggered immune response, suppressed follicle development and luteinization, imposing metabolism dysfunction and hormone imbalance in ovary. Further, trends of phenotypical infertility symptoms and endocrine changes in underweight female patients mirrored the alterations observed in our leptin pigs. These findings suggest that leptin-overexpressing pigs could serve as a valuable animal model for studying infertility and investigating potential therapeutic interventions. Overall design: Leptin overexpressed pig (C013) was re-cloned and the cloned offspring (?) were mated with the Duroc/Landrace/Yorkshire sow (DLY, ?) to obtain the F1 generation, and then the F1 generation was further crossed to obtain the F2 generation. The offspring (F2-generation) with leptin overexpression were then screened out by PCR, qPCR, and immunoblotting, etc. The body weight, body size and estrus of leptin overexpressed sows and control group were monitored. The changes in body weight and body size were plotted, and the pubertal onset and inter-estrous interval were calculated. The breeding efficiency of leptin overexpressed sows was explored by mating with fertile male and pregnancy was monitored. The serum profile of reproductive hormones was evaluated using radio immune assay (RIA) and enzyme linked immunosorbent assay (ELISA). Next, the ovaries of leptin and control sows were collected and protein abundance and mRNA expression of steroidogenic enzymes and apoptotic markers were evaluated at tissue and cell levels. Meanwhile, the back fat thickness of leptin overexpressed sows was measured by ultrasonography and serum levels of circulating fatty acids were measured by gas chromatography. Finally, the ovarian samples were sent for the transcriptomic profiling.