The Golgi Glycoprotein MGAT4D is an Intrinsic Protector of Testicular Germ Cells From Mild Heat Stress

Abstract

Male germ cells are sensitive to heat stress and testes must be maintained outside the body for optimal fertility. However, no germ cell intrinsic mechanism that protects from heat has been reported. Here, we identify the germ cell specific Golgi glycoprotein MGAT4D as a protector of male germ cells from heat stress. Mgat4d is highly expressed in spermatocytes and spermatids. Unexpectedly, when the Mgat4d gene was inactivated globally or conditionally in spermatogonia, or mis-expressed in spermatogonia, spermatocytes or spermatids, neither spermatogenesis nor fertility were affected. On the other hand, when males were subjected to mild heat stress of the testis (43 °C for 25 min), germ cells with inactivated Mgat4d were markedly more sensitive to the effects of heat stress, and transgenic mice expressing Mgat4d were partially protected from heat stress. Germ cells lacking Mgat4d generally mounted a similar heat shock response to control germ cells, but could not maintain that response. Several pathways activated by heat stress in wild type were induced to a lesser extent in Mgat4d[−/−] heat-stressed germ cells (NFκB response, TNF and TGFβ signaling, Hif1α and Myc genes). Thus, the Golgi glycoprotein MGAT4D is a novel, intrinsic protector of male germ cells from heat stress. MGAT4D is designated family member D of the MGAT4 gene family by the Human Genome Nomenclature Committee based on sequence similarity to other members, including MGAT4A and MGAT4B. The latter are N-acetylglucosaminyltransferases (GlcNAcTs) that add a β1, 4GlcNAc to complex N-glycans. However, when MGAT4D is transfected into cultured cells, it does not appear to have GlcNAcT activity. Rather, it inhibits MGAT1 activity, the GlcNAcT responsible for initiating complex N-glycan synthesis 1. Because of this inhibitory activity, the protein was termed GnT1IP for GlcNAcT1 Inhibitory Protein. The Mgat4d gene is highly expressed in mouse testis with little expression in other mouse tissues 2. Based on RNA-seq analysis, it is expressed in spermatocytes and spermatids, but not in spermatogonia, sperm or Sertoli cells 3. MGAT4D is the most abundant protein in purified Golgi from rat testis germ cells 4. Characterization of the interactions of MGAT4D in the Golgi using a fluorescence resonance energy transfer (FRET) assay showed that it interacts with MGAT1 but not MGAT2, MGAT3, MGAT4B or MGAT5 3. Since knockout of Mgat1 in spermatogonia disrupts spermatogenesis and results in infertility 5,6 , deletion or overexpression of Mgat4d in germ cells were both expected to have effects on spermatogenesis. In this paper, we show that unexpectedly, deletion of Mgat4d globally, or specifically in spermatogonia, or mis-expression of Mgat4d in spermatogonia, spermatocytes or spermatids, do not appear to alter spermatogenesis in young or aged mice, and do not affect fertility. However, mild heat stress of the testis in aged mice revealed that germ cells lacking Mgat4d exhibited more damage and apoptosis following heat stress. By contrast, a Mgat4d transgene expressed i...

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