Signaling Enzymes Required for Sperm Maturation and Fertilization in Mammals

Abstract

In mammals, motility and fertilizing ability of spermatozoa develop during their passage through the epididymis. After ejaculation, sperm undergo capacitation and hyperactivation in the female reproductive tract - a motility transition that is required for sperm penetration of the egg. Both epididymal initiation of sperm motility and hyperactivation are essential for male fertility. Motility initiation in the epididymis and sperm hyperactivation involve changes in metabolism, cAMP (cyclic adenosine mono-phosphate), calcium and pH acting through protein kinases and phosphatases. Despite this knowledge, we still do not understand, in biochemical terms, how sperm acquire motility in the epididymis and how motility is altered in the female reproductive tract. Recent data show that the sperm specific protein phosphatase PP1γ2, glycogen synthase kinase 3 (GSK3), and the calcium regulated phosphatase calcineurin (PP2B), are involved in epididymal sperm maturation. The protein phosphatase PP1γ2 is present only in testis and sperm in mammals. PP1γ2 has a isoform-specific requirement for normal function of mammalian sperm. Sperm PP1γ2 is regulated by three proteins - inhibitor 2, inhibitor 3 and SDS22. Changes in phosphorylation of these three inhibitors and their binding to PP1γ2 are involved in initiation and activation of sperm motility. The inhibitors are phosphorylated by protein kinases, one of which is GSK3. The isoform GSK3α is essential for epididymal sperm maturation and fertility. Calcium levels dramatically decrease during sperm maturation and initiation of motility suggesting that the calcium activated sperm phosphatase (PP2B) activity also decreases. Loss of PP2B results in male infertility due to impaired sperm maturation in the epididymis. Thus the three signaling enzymes PP1γ2, GSK3, and PP2B along with the documented PKA (protein kinase A) have key roles in sperm maturation and hyperactivation. Significantly, all these four signaling enzymes are present as specific isoforms only in placental mammals, a testimony to their essential roles in the unique aspects of sperm function in mammals. These findings should lead to a better biochemical understanding of the basis of male infertility and should lead to novel approaches to a male contraception and managed reproduction.

Keywords: GSK3α; PKA; PP1γ2; PP2B; contraception; epididymal sperm maturation; fertility; hyperactivation.

FIGURE 1

Schematic diagram showing some of the signaling events during sperm hyperactivation. CatSper channels are activated either by progesterone or alkalization of sperm cytosol to permit calcium entry. The Cl^––HCO₃^– exchanger, SLC26A3 and the Na⁺–HCO₃^– co-transporter, and Na⁺–H⁺ exchanger, NHE1 (Slca9c), aid in alkalization of sperm pHi. Slo3 K⁺-channels are activated by alkalization and causes hyperpolarization. Both Ca²⁺ and HCO₃^– stimulate sperm sAC to increase cAMP levels. Cyclic AMP, in turn activates sperm PKA (PRKA), which is expected to phosphorylate several substrates including protein tyrosine kinases (PTKs). Ca²⁺ also activates calmodulin (CAM) and calmodulin dependent phosphatase, calcineurin (PP2B) and a protein kinase CAMKII. The downstream effect and mechanism of action of these enzymes in promoting sperm fertilization is not well understood. Index: dotted arrow indicates activation, upward arrow denotes increase, and question mark (?) indicates unknown mechanism.

FIGURE 2

(A) Generation of PP1γ isoforms. The Ppp1cc contains 8 exons and 7 introns. The Pp1γ1 mature mRNA (2.3 kb) contains exons 1 through 7. Intron 7 is retained as an extended exon leading to the eight amino acid C-terminus of PP1γ1 (note that “exon 8” is part of its 3′UTR). The PP1γ1 encodes a protein containing 323 amino acids derived from the seven exons along with the 8 amino acid C-terminus from the extended exon 7. In the post-meiotic germ cells in testis the intron 7 is spliced out, thus, producing a shorter Pp1γ2 transcript of approximately 1.7 kb. Exon 8 codes for the 22 amino acid C-terminus in PP1γ2. Thus, the amino acid sequences of PP1γ1 and PP1γ2 are identical in all respects except for their extreme C-termini. (B) Constructs for generating transgenic PP1γ1 mice. Rescues I–III constructs contain the entire or a portion of intron 7 which is part of the 3′UTR of the messenger RNA for PP1γ1. There was little or no transgenic expression of PP1γ1 in testis of mice generated from these constructs. The last rescue construct (Rescue IV) lacks the 0.9 kb region of intron 7 following the stop codon in PP1γ1 mRNA. Transcript from this construct will resemble PP1γ2 mRNA except that PP1γ1 protein will be produced. The transgenic mice produced from this construct expressed high testis levels of transgenic PP1γ1 and rescued spermatogenesis but not sperm fertility.

FIGURE 3

(A–B) Regulation of PP1γ2 activity by I3, SDS22 and I2. (A) I3 and SDS bound PP1γ2 is catalytically inactive. Binding of these inhibitors to PP1γ2 is altered in caudal compared to caput sperm and in sperm from infertile transgenic KO mice (see text). (B) Inhibitor 2, a regulator of PP1γ2, is known to be phosphorylated by GSK3. In caput but not in caudal epididymal sperm I2 is expected to be phosphorylated due to changes in GSK3 activity. This is one of the ways by which catalytic activity of PP1γ2 is thought to decrease during epididymal sperm maturation. (C) Schematic diagram of the proposed interrelationship between GSK3α, PKA Cα2, PP1γ2 and PP2B (PPP3R2/CC) during epididymal sperm maturation and sperm hyperactivation in female reproductive tract. Curved orange arrow(s) indicate relative degree of activities of the enzymes; straight black arrow(s) denote relative level of the ion/protein inside the cell.

FIGURE 4

(A) Conservation of C-terminus of PPP1CC2 in mammals. The 5′ and 3′ splice sites for the generation of PP1γ2 in Ppp1cc is found only in mammals. These splice sites and a recognizable exon 8 are absent in Ppp1cc in the genomes of the duck billed platypus and the non-mammalian vertebrates. The C-terminal 22 amino acid residues (316–337) are virtually identical in all placental mammals for which annotated genomic data bases exist. Sequences from a few of these mammals are shown above. (B) PKA catalytic subunit in somatic cells (Cα1) and sperm (Cα2) are derived from alternate exons 1a and 1b. Cα1 is present in somatic cell. Cα2 produced in testis using exon 1b is present only in mammals. Exon 1b is present and conserved in all placental mammals. (C) Conservation of GSK3α isoform in mammals. The glycine rich N-terminus of the GSK3 alpha isoform is conserved in placental mammalian species, but not in non-mammalian vertebrates. (D) Conservation of testis-specific isoform of PP2B, PPP3R2 (regulatory subunit) across different mammalian species. While the entire PPP3R2 is present and conserved in all mammals only a portion of the N-terminus sequence of PPP3R2 is shown.

FIGURE 5

A simplified summary of spatial and temporal organization of GSK3α, PKA Cα2, PP1γ2, and PP2B (PPP3R2/CC). Upper panel image was adopted and modified from Dey et al. (2018, ; it shows localization of these enzymes in different compartments of a sperm cell; all of these four enzyme isoforms co-localizes only in sperm midpiece. Lower panel demonstrates how these enzymes regulate sperm functions starting from its synthesis (i.e., spermatogenesis) till fertilization. (↑) and (⊤) indicate stimulatory and inhibitory effect, respectively.