Scientists have transplanted testicular cells from the same mice, which were sterile due to the lack of gene Cldn11 (encodes a protein of tight junctions of the blood-testicular barrier) and in the seminiferous tubules began to develop functional gametes. As a result of fertilization of oocytes with spermatozoa of these mice developed 11 mice. Perhaps the reason for fertility restoration was the change in the balance between the different proteins of tight junctions of the blood-testicular barrier, but the exact mechanism is still unclear. Article published in the journal Proceedings of the National Academy of Sciences.
Hemato-testicular barrier is formed between the blood vessels and the seminiferous tubules and protects the emerging sperm from the immune system. The basis of the barrier — Sertoli cells, the processes which form between close contacts and share the space of seminiferous tubules on basal part, where the stem cells are okolovrastno, where developing sperm. After a stem cell (spermatogonium) divides by mitosis several times, it turns out a clone of several connected bridges of spermatocytes. The spermatocytes migrate through the blood-testicular barrier in oculoplasty part and enter into meiosis.
If the hemato-testicular barrier becomes permeable, meiosis of spermatocytes disrupted, resulting in sterility. This can occur if there is not enough proteins that form tight contacts between Sertoli cells. Such proteins there are in the spermatogonia and spermatocytes, although these cells contain tight junctions. The role of tight junctions proteins in stem cells remains to be seen.
Scientists from Japan, under the leadership of MiTo Kanatsu-Shinohara (Mito Kanatsu-Shinohara) from Kyoto University investigated the testes of sterile mice by gene nokauti of claudina CLDN11 protein of tight junctions. Sections of the testes of 11-month-old mice immunohistochemically stained for various markers. In particular we studied the expression of apoptosis markers to know which cells die when you turn off Cldn11.
Then spermatogonia destroyed by busulfan, which inhibits the work of germ cells, but does not pass through the blood-testicular barrier. The scientists looked at how the lack of proteins of tight junctions on other cells can affect the Sertoli cells.
To test whether the stem cells of the testes without CLDN11 to differentiate in a normal microenvironment, spermatogonia nokauti for Cldn11 and control mice (donor cells produced green fluorescent protein) were transplanted into the animals, spermatogonia which had been destroyed by busulfan. Then from the same animals transplanted primary stem cells.
Finally, spermatogonia transplanted from a healthy green mice (transgenic animals, whose cells isolated green fluorescent protein) nokauti for Cldn11 individuals. To find out whether the absence of Cldn11 colonization of spermatogonia in the testes, the blood-testicular barrier which has been formed by scientists using RNAi turned off this gene in Mature wild type mice (this procedure is called a knockdown).
Have nokauti for Cldn11 mice testicle size was smaller than wild-type animals; in the epididymis and in the testes was not Mature spermatozoa, although in the seminiferous tubules found sex cells. Dramaticheskih the number of germ cells outside of the blood-testicular barrier was reduced (p < 0.05). The expression of markers of apoptosis were increased in spermatocytes but not in spermatogonia.