Kula N. Jha

Evidence of the presence of calcium/calmodulin-dependent protein kinase IV in human sperm and its involvement in motility regulation

The mechanisms involved in the regulation of mammalian sperm motility are not well understood. Calcium ions (Ca2+) have been suggested to play a key role in the maintenance of motility; nevertheless, how Ca2+ modulates this process has not yet been completely characterized. Ca2+ can bind to calmodulin and this complex regulates the activity of multiple enzymes, including Ca2+/calmodulin-dependent protein kinases (CaM kinases). Results from this study confirmed that the presence of Ca2+ in the incubation medium is essential for maintaining human sperm motility. The involvement of CaM kinases in Ca2+ regulation of human sperm motility was evaluated using specific inhibitors (KN62 and KN93) or their inactive analogues (KN04 and KN92 respectively). Sperm incubation in the presence of KN62 or KN93 led to a progressive decrease in the percentage of motile cells; in particular, incubation with KN62 also reduced sperm motility parameters. These inhibitors did not alter sperm viability, protein tyrosine phosphorylation or the follicular fluid-induced acrosome reaction; however, KN62 decreased the total amount of ATP in human sperm. Immunological studies showed that Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is present and localizes to the human sperm flagellum. Moreover, CaMKIV activity increases during capacitation and is inhibited in the presence of KN62. This report is the first to demonstrate the presence of CaMKIV in mammalian sperm and suggests the involvement of this kinase in the regulation of human sperm motility.

Biochemical and Structural Characterization of Apolipoprotein A-I Binding Protein, a Novel Phosphoprotein with a Potential Role in Sperm Capacitation

The physiological changes that sperm undergo in the female reproductive tract rendering them fertilization-competent constitute the phenomenon of capacitation. Cholesterol efflux from the sperm surface and protein kinase A (PKA)-dependent phosphorylation play major regulatory roles in capacitation, but the link between these two phenomena is unknown. We report that apolipoprotein A-I binding protein (AI-BP) is phosphorylated downstream to PKA activation, localizes to both sperm head and tail domains, and is released from the sperm into the media during in vitro capacitation. AI-BP interacts with apolipoprotein A-I, the component of high-density lipoprotein involved in cholesterol transport. The crystal structure demonstrates that the subunit of the AI-BP homodimer has a Rossmann-like fold. The protein surface has a large two compartment cavity lined with conserved residues. This cavity is likely to constitute an active site, suggesting that AI-BP functions as an enzyme. The presence of AI-BP in sperm, its phosphorylation by PKA, and its release during capacitation suggest that AI-BP plays an important role in capacitation possibly providing a link between protein phosphorylation and cholesterol efflux.

Identification of unknown protein function using metabolite cocktail screening.

Proteins of unknown function comprise a significant fraction of sequenced genomes. Defining the roles of these proteins is vital to understanding cellular processes. Here, we describe a method to determine a protein function based on the identification of its natural ligand(s) by the crystallographic screening of the binding of a metabolite library, followed by a focused search in the metabolic space. The method was applied to two protein families with unknown function, PF01256 and YjeF_N. The PF01256 proteins, represented by YxkO from Bacillus subtilis and the C-terminal domain of Tm0922 from Thermotoga maritima, were shown to catalyze ADP/ATP-dependent NAD(P)H-hydrate dehydratation, a previously described orphan activity. The YjeF_N proteins, represented by mouse apolipoprotein A-I binding protein and the N-terminal domain of Tm0922, were found to interact with an adenosine diphosphoribose-related substrate and likely serve as ADP-ribosyltransferases. Crystallographic screening of metabolites serves as an efficient tool in functional analyses of uncharacterized proteins.

TSKS concentrates in spermatid centrioles during flagellogenesis.

Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 and 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of Tssk1 and 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.

Germ cell‐specific disruption of the Meig1 gene causes impaired spermiogenesis in mice

Meiosis expressed gene 1 (Meig1) was originally identified in a search for mammalian genes potentially involved in meiosis. Seven mouse Meig1 transcripts with the same coding region, but different 5′‐UTRs, have been identified. These transcripts have different tissue distributions, two are only present in the testis. In the testis, Meig1 is present in germ cells and Sertoli cells. A Meig1 conditional knockout model has been generated. When Meig1 was inactivated globally by crossing with Cmv‐Cre transgenic mice, the Meig1‐deficient males were sterile due to severe spermiogenic defects, and had no obvious defects in meiosis. To further study its role in individual cell types in the testis, the Meig1flox mice were crossed with Hsp2a‐Cre, Prm‐Cre, and Amh‐Cre mice, in which the Cre recombinase is driven by the heat shock protein 2 (Hsp2a) gene promoter (expressed in spermatocytes), the protamine 1 gene promoter (expressed in post‐meiotic spermatids) and the anti‐Mullerian hormone (Amh) gene promoter (expressed in Sertoli cells) respectively. Both Meig1 mRNA and protein were undetectable in testis of the Hsp2a‐Cre; Meig1flox/flox mice and all the mutant adult males tested were sterile. This phenotype mirrors that of the Cmv‐Cre; Meig1flox/flox mice. Even though the total testicular Meig1 mRNA and protein expression levels were dramatically reduced in testis of the Prm‐Cre; Meig1flox/flox males, all the mice tested were fertile, and there was no significant difference in sperm count and sperm motility compared with age‐matched Meig1flox/flox male mice. Disruption of Meig1 in the Sertoli cells did not affect the MEIG1 protein expression. Amh‐Cre; Meig1flox/flox males were fertile, and produced the same amount of spermatozoa as age‐matched Meig1flox/flox mice. The testicular histology was also normal. Our results indicate that MEIG1 regulates spermiogenesis through effects in germ cells alone, and that the Meig1 gene must be active during a discrete period in spermatogenesis after which it is dispensable.