Understanding the molecular basis of sperm capacitation through kinase design.

T hirty years ago, on July 25, Steptoe and Edwards reported the birth of Louise Joy Brown, the first successful ‘‘Test-Tube’’ baby (1). This achievement followed a lack of success of in vitro fertilization experiments for almost 80 years since the first attempts in 1878. These early failures were due mainly to a lack of comprehension of sperm physiology. In the early 1950s, Chang (2) and Austin (3) demonstrated independently that sperm had to be in the female reproductive tract for a finite period before acquiring fertilizing capacity. This phenomenon is known as sperm capacitation. What made this finding a necessary step for the consequent development of in vitro fertilization was the understanding that certain factors in the female were needed for the sperm to become fertile. A logical follow-up of the discovery of sperm capacitation occurred some years later when Chang demonstrated mammalian in vitro fertilization conclusively by showing that eggs from a black rabbit fertilized in vitro by capacitated sperm from a black male, and transferred to a white female, resulted in the birth of a litter of black offspring (4). In vitro fertilization was made possible by the discovery of sperm capacitation and raised the interest to study the molecular basis of this process. Inherent to these studies, capacitation was defined as the physiological changes occurring in the female reproductive tract that render the sperm able to fertilize. These changes involved a series of sequential and parallel processes; some of them take place as soon as the sperm is ejaculated, whereas others arise over a longer period in the female tract or in a medium that supports in vitro capacitation. Interestingly, both early and late events are centrally regulated by protein kinase A (PKA). The work by Morgan et al. (5) in a recent issue of PNAS takes a chemical-genetic switch approach to understand the temporal action of this enzyme in sperm capacitation. Regarding this approach, to facilitate consideration of the complex cascade of molecular events that occur during capacitation, a discussion of this process may be divided into fast and slow capacitation events (Fig. 1). A very early event in sperm capacitation is the activation of sperm motility. Although sperm stored in the cauda epididymis consume oxygen at a high rate, they are immotile. The vigorous movement of the flagellum starts immediately after sperm are released from the epididymis and come into contact with high HCO3 and Ca2 concentrations present in the seminal f luid. The transmembrane movement of HCO3 has been associated with the increase in intracellular pH (pHi) observed during capacitation. However, another likely target for HCO3 as well as Ca2 actions in sperm is the regulation of cAMP metabolism through stimulation of a unique type of adenylyl cyclase (SACY). In contrast to transmembrane adenylyl cyclases, SACY does not respond to activators of Gs, the G protein stimulator of transmembrane cyclases, such as cholera toxin or nonhydrolyzable analogs of GTP (e.g., GTP S). Furthermore, SACY is stimulated by HCO3 anions and multiple evidences indicate that this atypical cylase is the main HCO3 target during sperm capacitation. As a consequence of SACY activation, intracellular levels of cAMP increase and activate PKA. Once activated, PKA phosphorylates various target proteins that are presumed to initiate several signaling pathways. In sperm exposed to HCO3 , cAMP rises to a maximum within 60 sec, and the increase in PKA-dependent phosphorylation begins within 90 sec (for review, see ref. 6). In contrast to the fast activation of sperm motility, other capacitation-associated processes require longer incubation periods. These slower processes can be accomplished in vitro by using sperm incubated in defined media. In all cases, in vitro capacitation media contain a protein source that usually is BSA; and an assortment of ions including HCO3 and Ca2 . The mechanisms by which these media components are able to