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A major change in the transcriptome of testicular Sertoli cells (Scs) at the onset of puberty enables them to induce robust spermatogenesis. Through comprehensive literature mining, we generated a list of genes crucial for Sc functioning and computationally predicted the microRNAs regulating them. Differential expression analysis of microRNAs in infant and pubertal rat Scs showed that miR-382-3p levels decline significantly in pubertal Scs. Interestingly, miR-382-3p was found to regulate genes like Ar and Wt1, which are crucial for functional competence of Scs. We generated a transgenic (Tg) mouse model in which pubertal decline of miR-382-3p was prevented by its overexpression in pubertal Scs. Elevated miR-382-3p restricted the functional maturation of Scs at puberty, leading to infertility. Prevention of decline in miR-382-3p expression in pubertal Scs was responsible for defective blood-testis barrier (BTB) formation, severe testicular defects, low epididymal sperm counts and loss of fertility in these mice. This provided substantial evidence that decline in levels of miR-382-3p at puberty is the essential trigger for onset of robust spermatogenesis at puberty. Hence, sustained high levels of miR-382-3p in pubertal Scs could be one of the underlying causes of idiopathic male infertility and should be considered for diagnosis and treatment of infertility.

Although rats are preferred over mice as an animal model, transgenic animals are generated predominantly using mouse embryos. There are limitations in the generation of transgenic rat by embryo manipulation. Unlike mouse embryos, most of the rat embryos do not survive after male pronuclear DNA injection which reduces the efficiency of generation of transgenic rat by this method. More importantly, this method requires hundreds of eggs collected by killing several females for insertion of transgene to generate transgenic rat. To this end, we developed a noninvasive and deathless technique for generation of transgenic rats by integrating transgene into the genome of the spermatogonial cells by testicular injection of DNA followed by electroporation. After standardization of this technique using EGFP as a transgene, a transgenic disease model displaying alpha thalassemia was successfully generated using rats. This efficient method will ease the generation of transgenic rats without killing the lives of rats while simultaneously reducing the number of rats used for generation of transgenic animal.

Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools, but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious, and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique.