A study to assess the assembly of a functional blood-testis barrier in developing rat testes

The blood-testis barrier (BTB) is an important ultrastructure in the seminiferous tubule of the mammalian testis that segregates the events of spermatogenesis, in particular post-meiotic germ cell development, from the harmful substances in the environment including toxicants and drugs, as well as from the unwanted hormones and biomolecules in the systemic circulation. It is known that the BTB is assembled by ∼15-21 days postpartum (dpp) in rats coinciding with the onset of late cell cycle progression, namely the formation of zygotene and pachytene spermatocytes by day 15-18 dpp. This is to prepare for: (1) the differentiation/transformation of pachytene spermatocytes to diplotene and dictyate spermatocytes and (2) meiosis I and II, which take place by 23-26 and 26 dpp, respectively. Recent findings have shown spermatogonia/spermatogonial stem cells (SSC) in the tubules failed to re-initiate spermatogenesis by differentiating spermatogonia beyond type A spermatogonia in the absence of a functional BTB, leading to meiotic arrest. These studies thus illustrate that a functional BTB is crucial to the initiation and/or re-initiation of spermatogenesis. Herein, we sought to examine the precise time window when a functional and intact BTB is established in the developing rat testis during the final stage of cell cycle progression and meiosis. Using the techniques of: (1) dual-labeled immunofluorescence analysis to assess the distribution of integrated proteins at the tight junction (TJ), basal ectoplasmic specialization [basal ES, a testis-specific atypical adherens junction (AJ) type] and gap junction (GJ) at the BTB, (2) functional assay to assess the BTB integrity in vivo, (3) immunoblot analysis to monitor changes in steady-state levels of adhesion proteins at the BTB, and (4) co-immunoprecipitation to assess changes in protein-protein interactions at the BTB, it was shown that a BTB was being assembled by day 15-20 dpp, but a functional BTB was not fully established until day 25 dpp in Sprague-Dawley rats, tightly associated with the onset of meiosis I and II. These findings thus illustrate the significance of the BTB on cell cycle progression and the preparation for meiosis, such as germ cell differentiation beyond type A spermatogonia.