Complexin I is required for mammalian sperm acrosomal exocytosis

Abstract

Regulated exocytosis in many cells is controlled by the SNARE complex, whose core includes three proteins that promote membrane fusion. Complexins I and II are highly related cytosolic proteins that bind tightly to the assembled SNARE complex and regulate neuronal exocytosis. Like somatic cells, sperm undergo regulated exocytosis; however, sperm release a single large vesicle, the acrosome, whose release has different characteristics than neuronal exocytosis. Acrosomal release is triggered upon sperm adhesion to the mammalian egg extracellular matrix (zona pellucida) to allow penetration of the egg coat. Membrane fusion occurs at multiple points within the acrosome but how fusion is activated and the formation and progression of fusion points is synchronized is unclear. We show that complexins I and II are found in acrosome-intact mature sperm, bind to SNARE complex proteins, and are not detected in sperm after acrosomal exocytosis (acrosome reaction). Although complexin-I-deficient sperm acrosome-react in response to calcium ionophore, they do not acrosome-react in response to egg zona pellucida proteins and have reduced fertilizing ability, in vitro. Complexin II is present in the complexin-I-deficient sperm and its expression is increased in complexin-I-deficient testes. Therefore, complexin I functions in exocytosis in two related but morphologically distinct secretory processes. Sperm are unusual because they express both complexins I and II but have a unique and specific requirement for complexin I.

Figure 1

Complexins I and II are expressed in mouse testis and the proteins are found in mature acrosome-intact sperm. (A) Complexin RT-PCR. Primers were designed to amplify the entire complexin I and complexin II cDNAs (cpx 1 and 2). Lanes were loaded as follows: oligo dT-primed brain cDNA (1), random-primed brain cDNA (2), brain RNA (3), oligo dT-primed testis cDNA (4), random-primed testis cDNA (5), testis RNA (6), and water negative control (7). Complexin I primers amplified a 509 bp product from testis and brain cDNA. Complexin II primers amplified a 483 bp product from both brain and testis cDNA. Negative controls were RNA without reverse transcriptase or water (no cDNA). (B) Complexin western blot. Antibodies to complexins I and II detected the 18 kDa complexin I and 19 kDa complexin II proteins in mouse brain (B, 1 μg protein loaded), mouse testis (T, 60 μg protein loaded), and mouse cauda epididymal sperm (S, 80 μg protein loaded). Both complexins were much less abundant in extracts from testis and sperm compared to brain. The antibodies non-specifically bound to proteins larger than 25 kDa. These results are representative of more than 5 experiments.

Figure 2

Complexins I and II are found in acrosome-intact but not acrosome-reacted sperm. Mouse sperm were incubated at 37°C for 60 min for capacitation and the acrosome reaction was induced by calcium ionophore A23187 for 60 min. Coomassie Blue staining to show acrosomal status in control (A, uncapacitated) and A23187-treated (acrosome-reacted) sperm (B). (C) Western Blots show complexins were not observed in sperm following the acrosome reaction. Proteins from the same number of control (NON-AR) and A23187-treated sperm (AR) were loaded for Western blots. Complexins were not detected in A23187-treated sperm. These results are representative of three experiments.

Figure 3

Sperm from complexin I deficient mice are unable to acrosome react in response to zona pellucida proteins and display reduced fertility in vitro. (A) Sperm from complexin I deficient mice (KO) and wild type (WT) animals of the same age were incubated with soluble mouse zona pellucida proteins to induce the acrosome reaction. Fluorescein isothiocyanate-labeled Pisum sativum agglutinin (FITC-PSA) was used to detect acrosomes. Means and s.e.m. of five experiments performed in duplicate are shown. The asterisk denotes that wild type sperm responded to zona pellucida proteins with acrosome reactions, as assessed by the Student t test (p < 0.05). (B) Sperm from complexin I deficient mice (KO) and wild type (WT) animals of the same age were incubated with 10 μM calcium ionophore A23187. After 60 min, aliquots of sperm were fixed and stained with FITC-PSA to detect acrosomes. Means and s.e.m. of three experiments performed in triplicate are shown. Asterisks indicate that sperm from both WT and KO underwent acrosome reactions induced by calcium ionophore (Student t test, P < 0.05) but the frequency was not different between sperm from WT and complexin I KO mice. (C) Mouse oocytes were co-incubated with 1 × 10⁴/ml or 1 × 10⁵/ml of sperm from complexin I deficient mice or wild type mice for 42 hours. After co-incubation, embryos and oocytes were fixed and stained with propidium iodide to detect nuclei. The presence of two-cell embryos or two pronuclei or polar bodies was considered as evidence of fertilization. Results are means and s.e.m. of five experiments.

Figure 4

Complexin II expression is upregulated in complexin I deficient mice. Complexin antibodies detected the 18 kD complexin I and 19 kD complexin II proteins in wild type mouse brain (B, 1 μg protein), wild type mouse testes (T, +/+; 60 μg protein); but only detect 19 kD complexin II in testis tissue from complexin I heterozygote mice (T, +/-; 20 μg protein) and homozygote null mice (T, -/-; 20 μg protein). The abundance of complexin II was increased when complexin I was deficient in the testis. This result represents three experiments.

Figure 5

Complexins I and II bind a 120-kDa protein complex. Mouse sperm protein (3.3 mg) was incubated with purified GST protein (50 μg), or recombinant GST-complexin I (50 μg) or GST-complexin II (50 μg). Bound material was separated and bands identified by SDS-PAGE (samples were not boiled) and Coomassie Blue staining. The asterisks show the 120-kDa protein complex that interacts specifically with complexin I or II. Each band was excised and analyzed by mass spectrometry. The numbers on the left correspond to the migration of standards loaded in the outside lanes. Similar SDS-PAGE results were obtained in two experiments.