Stanley Meizel

Steroid induced exocytosis: The human sperm acrosome reaction

By a combination of organic precipitation and high pressure liquid chromatography, human sperm acrosome reaction inducing activity has been purified from the fluid aspirated from preovulatory human ovarian follicles and identified as 4-pregnen-3,20-dione (progesterone) and 4-pregnen-17 alpha-ol-3,20-dione (17 alpha-hydroxyprogesterone). It is argued that progesterone is present at the site of fertilization of placental mammals in concentrations sufficient for activity, and hence provides a mechanism of inducing the acrosome reaction, an exocytotic event, in vivo.

Progesterone acts at the plasma membrane of human sperm

There has been increasing interest in the relationship between rapid effects of steroids and steroid-plasma membrane interaction. This laboratory has previously reported that progesterone increases human sperm cytosolic free calcium ([Ca2+]i) and thereby initiates the human sperm acrosome reaction (AR) in less than 1 min. Herein, to test whether progesterone acts at the sperm plasma membrane, progesterone 3-(O-carboxymethyl)oxime: bovine serum albumin (BSA) conjugate (free of unconjugated progesterone) was added to capacitated human sperm. Fura-2 assays were used to detect less than 1 min changes in [Ca2+]i, and indirect immunofluorescence was used to assay the AR occurring 1 min after stimulus addition. The conjugate increased [Ca2+]i and the AR (though less than did unconjugated progesterone). Enzyme immunoassays demonstrated that the concentrations of unconjugated progesterone in conjugate-treated sperm suspensions did not increase over those of control suspensions. Since the progesterone: BSA conjugate presumably does not cross the sperm plasma membrane, progesterone must act at that membrane to increase [Ca2+]i and the AR.

THE IMPORTANCE OF HYDROLYTIC ENZYMES TO AN EXOCYTOTIC EVENT, THE MAMMALIAN SPERM ACROSOME REACTION

The mammalian sperm acrosome reaction is a unique form of exocytosis, which includes the loss of the involved membranes. Other laboratories have suggested the involvement of hydrolytic enzymes in somatic cell exocytosis and membrane fusion, and in the invertebrate sperm acrosome reaction, but there is no general agreement on such an involvement. Although reference was made to such work in this review, the focus of the review was on the evidence (summarized below) that supports or fails to support the importance of certain hydrolytic enzymes to the mammalian sperm acrosome reaction. Because the events of capacitation, the prerequisite for the mammalian acrosome reaction, and of the acrosome reaction itself are not fully understood or identified, it is not yet always possible to determine whether the role of a particular enzyme is in a very late step of capacitation or part of the acrosome reaction.

In vitro studies of the golden hamster sperm acrosome reaction: Completion on the zona pellucida and induction by homologous soluble zonae pellucidae☆

We have studied the occurrence of the golden hamster sperm acrosome reaction (AR) in vitro during interaction with the oocyte investments: the cumulus cell matrix and the zona pellucida. Hamster sperm were capacitated in a defined medium that does not induce the AR. These spermatozoa were allowed to interact with the ovum vestments, the events of which were recorded using high-speed videomicrography. Frame-by-frame analysis revealed that sperm did not complete the AR in the cumulus cell matrix, but did so on the zona pellucida. Furthermore, a higher percentage of sperm completed the AR on the zona pellucida of cumulus-invested than on cumulus-free eggs. We also investigated the effect of solubilized hamster and mouse zonae pellucidae on the hamster sperm AR. Addition of solubilized hamster zonae to capacitated sperm elicited the AR within 15 min. Solubilized mouse zonae were significantly less effective, indicating that the zona-induced AR in hamster sperm may be species specific. These results suggest that the hamster zona pellucida is an inducer of the AR in the intact or soluble form, and that the majority of spermatozoa traverse the cumulus cell matrix without completing the AR in our in vitro system.

TAURINE AND HYPOTAURINE: THEIR EFFECTS ON MOTILITY, CAPACITATION AND THE ACROSOME REACTION OF HAMSTER SPERM IN VITRO AND THEIR PRESENCE IN SPERM AND REPRODUCTIVE TRACT FLUIDS OF SEVERAL MAMMALS*

Previous work from this laboratory has shown that the β‐amino acid taurine can support and stimulate hamster sperm motility during in vitro capacitation in the presence or absence of epinephrine. The present report describes in vitro results which demonstrate that hypotaurine, a precursor of taurine, can also support and stimulate motility under these conditions and that a higher number of acrosome reactions occur in the presence of taurine as compared to hypotaurine (both in the presence and absence of epinephrine). In all cases, the greates percentage of acrosome reactions occurs in the presence of epinephrine. Whether these β‐amino acids act independently of epinephrine of in a synergistic manner with it remains to be determined. In addition to these in vitro studies, we report that hypotaurine and taurine are present at high levels in bovine follicular fluid, rabbit uterine and ampullar oviductal fluid (11 hr after mating, i.e., 1 hr after ovulation), monkey oviductal fluid, bovine adrenal cortex “motility factor” preparation and human, guinea pig and hamster sperm preparations. Based on these results, we suggest the possibility that taurine and hypotaurine may have roles in vivo in the maintenance and stimulation of sperm motility and stimulation of capacitation and/or acrosome reactions.

The sperm, a neuron with a tail: ‘neuronal’ receptors in mammalian sperm

A number of plasma membrane receptor types originally thought to be specific to neurons have been found in other somatic cells. More surprisingly, the mammalian sperm and neuron appear to share many of these‘neuronal’receptors. The morphology, chromosome number, genomic activity, and functions of those two cell types are as unlike as any two cells in the body, but they both achieve their highly disparate goals with the aid of a number of the same receptors. Exocytosis in neurons and sperm is essential to the functions of these cells and is strongly influenced by similar receptors.‘Neuronal’receptor types in sperm may also play a role in the control of sperm motility (a function of course not shared by neurons).

The pH of the hamster sperm acrosome.

The pH of the hamster sperm acrosome was estimated by a method based on the distribution of monoamines between membrane enclosed volumes maintaining pH gradients. A fluorescent amine, 9-aminoacridine, was used to permit both microscopic and fluorometric measurements of amine distribution. Cauda epididymal hamster sperm incubated with 9-aminoacridine accumulated the amine in the acrosomal volume. In the presence of NH4Cl or the ionophore Nigericin (compounds which discharge pH gradients) 9-aminoacridine fluorescence disappeared from the acrosome. Amine distribution between the acrosome and external volume was estimated by fluorometric measurement of sperm filtrates in the presence and absence of NH4Cl and Nigericin. These values, together with an estimated acrosomal volume of 0.4mu3 were used to calculate an acrosomal pH of less than 5. In addition, an acrosomal pH of 5 or less was obtained with 14C-methylamine. We suggest that such an acidic acrosomal pH of 5 or less could serve to inhibit the activation or autoactivation of the acrosomal zymogen proacrosin to acrosin, a trypsin-like enzyme involved in fertilization.

Involvement of Protein Kinase A and A Kinase Anchoring Protein in the Progesterone-Initiated Human Sperm Acrosome Reaction

Abstract The signal transduction pathways involved in the progesterone (P4)-initiated mammalian sperm acrosome reaction (AR) are not fully understood. To investigate the role of the protein kinase A (PKA) pathway in the P4-initiated AR, we probed this pathway by pretreating capacitated human sperm with reagents designed to either inhibit PKA activation or disrupt PKA/A kinase anchoring protein (AKAP) interactions. Preincubation with the stearated (membrane permeable) PKA inhibitor, PKI α 5-24 (S-PKI α 5-24), significantly inhibited the P4-initiated AR at 10 μM as compared to stearated control peptide. In contrast, preincubation with 100 μM nonstearated PKI α 5-24 did not significantly inhibit versus solvent control. Preincubation with the PKA inhibitor Rp-8-Br-cAMP at 500 μM and 150 μM significantly inhibited the P4-initiated AR versus 8-Br-cAMP and versus solvent. Preincubation with the anchoring inhibitory peptide S-Ht-31 significantly stimulated the P4-initiated AR at 10, 3, and 1 μM versus inactive control peptide. The stimulation of the P4-initiated AR by 3 μM S-Ht31 was significantly inhibited by the addition of 30 μM S-PKI α 5-24 prior to the addition of S-Ht31. Preincubation with S-PKI α 5-24 (30 μM) partially inhibited the ionomycin (50 μM)-initiated AR. A role for PKA in the P4-initiated AR may exist both upstream and downstream of Ca2+ entry. Our studies present the first evidence for the participation of PKA in the P4-initiated AR and also suggest that AKAPs are involved in the PKA-mediated events.

Regulation of intracellular pH in capacitated human spermatozoa by a Na+/H+ exchanger

We previously demonstrated that the progesterone‐ (P) initiated human sperm acrosome reaction (AR) was dependent on the presence of extracellular Na+ (Na+o). Moreover, Na+o depletion resulted in a decreased cytosolic pH (pHi), suggesting involvement of a Na+‐dependent pHi regulatory mechanism during the P‐initiated AR. We now report that the decreased pHi resulting from Na+o depletion is reversible and mediated by a Na+/H+ exchange (NHE) mechanism. To determine the role of an NHE in the regulation of pHi, capacitated spermatozoa were incubated in Na+‐deficient, bicarbonate/CO2‐buffered (0NaB) medium for 15–30 min, which resulted in an intracellular acidification as previously reported. These spermatozoa were then transferred to Na+‐containing, bicarbonate/CO2‐buffered (NaB) medium; Na+‐containing, Hepes‐buffered (NaH) medium; or maintained in the 0NaB medium. Included in the NaH medium was the NHE inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA). The steady‐state pHi was then determined by spectrofluorometric measurement of bis(carboxyethyl)‐5(6)‐carboxyfluoroscein (BCECF) fluorescence. EIPA (0.1 μM) significantly (P < 0.05) inhibited the pHi recovery produced by NaH medium. Moreover, the pHi in NaH medium was not significantly (P < 0.05) different than NaB medium. These results indicate that a Na+‐dependent, bicarbonate‐independent pHi regulatory mechanism, with a pharmacological characteristic consistent with an NHE, is present in capacitated spermatozoa. In support of the involvement of a sperm NHE, we also demonstrated specific immunoreactivity for a 100 kDa porcine sperm protein using an NHE‐1 specific monoclonal antibody. Interestingly, no significant (P = 0.79) effect was seen on the P‐initiated AR when EIPA was included in either the NaH or NaB medium. While these findings suggest that inhibition of NHE‐dependent pHi regulation in capacitated spermatozoa is not sufficient to block initiation of the AR by P, they do not preclude the possibility that an NHE mediates the regulation of capacitation or sperm motility. Mol. Reprod. Dev. 52:189–195, 1999.

Mice Deficient in CHRNA7, a Subunit of the Nicotinic Acetylcholine Receptor, Produce Sperm with Impaired Motility

Abstract In this study we investigate the role of the CHRNA7 subunit (also known as the alpha7 subunit) of the nicotinic acetylcholine receptor in mouse sperm function. We confirm by reverse-transcription-polymerase chain reaction the expression in adult mouse testis of Chrna7 mRNA and demonstrate the subunits presence in mouse sperm by immunoblot. Alpha-bungarotoxin binds a range of nicotinic acetylcholine receptor subunits, including the CHRNA7 subunit. Localization studies using a fluorescent alpha-bungarotoxin-tetramethyl-rhodamine conjugate revealed specific binding sites on the midpiece of mouse sperm with fainter alpha-bungarotoxin binding on the remainder of the flagellum. Mice engineered with a double-null disruption of the Chrna7 gene displayed only faint fluorescence on the midpiece, suggesting that the CHRNA7 contributed the majority of the observed alpha-bungarotoxin binding sites. The location of alpha-bungarotoxin binding suggested that nicotinic acetylcholine receptors may play an ionotropic role in sperm motility. Sperm from Chrna7−/− mice display no difference in number, morphology, viability or spontaneous acrosome reaction rate compared with Chrna7+/+ sperm. Studies using computer-assisted sperm analysis indicate the motility of Chrna7−/− sperm is significantly impaired. This impairment is characterized by significantly reduced swimming velocities, failure to maintain vigorous swimming, and lower levels of hyperactivated swimming patterns in Chrna7−/− sperm compared with Chrna7+/+ sperm. This is the first genetic evidence that sperm nicotinic acetylcholine receptors are important for maintenance of normal sperm motility.