Spermatozoa, as well as oocytes, are the only cells which can transmit genetic information to the next generation, and thus have specialized morphology and function for fertilization. However, genetic mechanisms that support the properties of spermatozoa are not fully understood. In addition, more than 10% of couples are experiencing infertility in western countries and Japan, and approximately half of the cause is attributed to males; however, the genetic cause of male infertility remains to be elucidated. Our laboratory, we are focusing to identify novel genes which are specifically expressed during sperm differentiation and to elucidate their molecular function or their physiological role in mice. Especially, we focus on membrane protein genes, because they play fundamental roles in cell and hold promise as drug target. Through these studies, we aim to understand the molecular basis for mammalian spermatogenesis and to apply the findings for elucidation of the cause of male infertility, efficient production of livestock, and population/fertility control of wildlife.
Recent findings in our laboratory are follows:
1) Slc22a14 plays a critical role for male fertility
We found that Slc22a14, a member of solute carrier (SLC) transporter family, plays a critical role for male fertility in mice. Mice lacking Slc22a14 gene shows severe male infertility. Although spermatogenesis in Slc22a14-/- mice are essentially normal, cauda epididymal spermatozoa shows reduced motility with abnormal flagellar bending. In addition, Slc22a14?deficient spermatozoa are also impaired in capacitation and ability of fertilization. We also found that the abnormal flagellar bending is caused by osmotic cell swelling and defect in annulus structure. It is plausible that deletion of Slc22a14 leads to change of osmotic pressure in spermatozoa; however, the molecule(s) that is transported by Slc22a14 is yet to be elucidated. We are investigating to identify the substrate of Slc22a14.
a. The mouse is a superior model animal to study the gene function at individual level, because manipulation of genome is relatively easy.
b. Morphology of cauda epididymal spermatozoa form wild-type (left) and Slc22a14-deficient mouse (right). Note that Slc22a14-/- sperm tails were mostly folded like hairpin (arrow) or V-shape (arrow head).
2) TMEM225 is a novel PP1g2 regulator localizing to the equatorial segment
We found that TMEM225 is specifically expressed in male germ cell and its expression increase during sperm differentiation in mice. In mature spermatozoa, TMEM225 is localized to the equatorial segment. We also revealed that TMEM225 directly bind to PP1g2, a sperm?specific isoform of protein phosphatase 1g (PP1g) via RVxF motif in C?terminal region and inhibit phosphatase activity. Since it has been demonstrated that activity of PP1g2 is necessary for normal spermiogenesis and relevant to capacitation and motility in spermatozoa, our results suggested that TMEM225 is involved in the differentiation and function of spermatozoa through the regulation of PP1g2 activity in mice.
Intracellular localization of TMEM225 in mouse sperm. TMEM225 (red) localized to the equatorial segment of the head. Sp56 (green) is a marker for anterior acrosome.
3) Identification of SAMT family protein as substrates of MARCH11
MARCH11 is spermatid?specific E3 ubiquitin ligase which localized to trans-Golgi network (TGN) and multivesicular bodies (MVB). Since ubiquitination act as a sorting signal of cargo proteins, MARCH11 has been postulated to regulate the selective protein sorting. However, the physiological substrate has not been identified. We found that SAMT family proteins, a novel 4-transmembrane protein family, are ubiquitinated by MARCH11 and delivered to lysosome through TGN-MVB pathway by ubiquitin-dependent manner. Proper protein sorting is necessary for normal morphogenesis during spermatogenesis. SAMTs may contribute to the normal morphogenesis of mouse sperms through the mediation of protein transport from the TGN to MVBs.
Expression and localization of SAMT1 in mouse testis. SAMT1 is expressed at Cap phase (C), early acrosome phase (EA), and late acrosome phase (LA), but not at Golgi phase (G) and maturation phase (M) of spermiogenesis (a). SAMT1 colocalize with GM130, a Golgi apparatus marker (b) and with MARCH11, a ubiquitin E3 ligase which localize to TGN and MVBs (c).