SEPT12/SPAG4/LAMINB1 Complexes Are Required for Maintaining the Integrity of the Nuclear Envelope in Postmeiotic Male Germ Cells

Abstract

Male infertility affects approximately 50% of all infertile couples. The male-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile or immature sperm, and sperm with structural defects such as those caused by premature chromosomal condensation and DNA damage. Our previous studies based on a knockout mice model indicated that SEPT12 proteins are critical for the terminal morphological formation of sperm. SEPT12 mutations in men result in teratozospermia and oligozospermia. In addition, the spermatozoa exhibit morphological defects of the head and tail, premature chromosomal condensation, and nuclear damage. However, the molecular functions of SEPT12 during spermatogenesis remain unclear. To determine the molecular functions of SEPT12, we applied a yeast 2-hybrid system to identify SEPT12 interactors. Seven proteins that interact with SEPT12 were identified: SEPT family proteins (SEPT4 and SEPT6), nuclear or nuclear membrane proteins (protamine 2, sperm-associated antigen 4, and NDC1 transmembrane nucleoproine), and sperm-related structural proteins (pericentriolar material 1 and obscurin-like 1). Sperm-associated antigen 4 (SPAG4; also known as SUN4) belongs to the SUN family of proteins and acts as a linker protein between nucleoskeleton and cytoskeleton proteins and localizes in the nuclear membrane. We determined that SEPT12 interacts with SPAG4 in a male germ cell line through coimmunoprecipitation. During human spermiogenesis, SEPT12 is colocalized with SPAG4 near the nuclear periphery in round spermatids and in the centrosome region in elongating spermatids. Furthermore, we observed that SEPT12/SPAG4/LAMINB1 formed complexes and were coexpressed in the nuclear periphery of round spermatids. In addition, mutated SEPT12, which was screened from an infertile man, affected the integration of these nuclear envelope complexes through coimmunoprecipitation. This was the first study that suggested that SEPT proteins link to the SUN/LAMIN complexes during the formation of nuclear envelopes and are involved in the development of postmeiotic germ cells.

SUN4 is essential for nuclear remodeling during mammalian spermiogenesis

“…The same is also true of SUN3 ( Figure 1A white arrow). The co-localization with lamin B1 is consistent with a recent report that the SUN4 N-terminal domain can form a complex with both lamin B1 and SEPT12, a GTP-binding protein (Yeh et al, 2015). This association implies that SUN4 could be localized at least in part to the INM.…”

SEPT12–NDC1 Complexes Are Required for Mammalian Spermiogenesis

“…NDC1 was identified as a SEPT12 interactor through the yeast 2-hybrid system by using a human testicular cDNA library [17]. In mice models, mutated NDC1 also affected mammalian spermatogenesis [25].…”

“…The SEPT12D197N (Asp197Asn; D197N) mutation has been isolated in infertile men with teratozoospermia and oligozoospermia [14,17]. In addition, knock-in mice with the SEPT12D197N mutation, which were used to mimic infertile men with SEPT12D197N, exhibited a high percentage of disorganized sperm annuli and necks [27].…”

“…Notably, this protein group contains (1) NDC1 transmembrane nucleoproteins (NDC1); (2) sperm-associated antigen 4 proteins (SPAG4); and (3) protamine-2 proteins (PRM2) [17]. PRM2 and PRM1 are major sperm proteins involved in nuclear packing [28], whereas SPAG4 (SUN4) belongs to the SUN family.…”

“…In humans, SEPT12 mutations in infertile men result in teratozoospermia and oligozoospermia [14,15,16]. In addition, several nuclear or nuclear membrane-related proteins were identified as SEPT12 interactors through the yeast 2-hybrid system; one of these interactors is NDC1 [17]. …”