ISOFORM-SPECIFIC ROLE OF PP1𝛾 2 IN SPERMFUNCTION AND MALE FERTILITY KINASE IN MAMMALS, A POTEINTAIL NON-HORMONAL CONTRACEPTIVE TARGETING EPIDIDYMAL SPERM
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2025-05
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Kent State University
Abstract
Abstract Part I: The protein phosphatase isoforms PP1γ1 and PP1γ2, alternate spliced products of the Ppp1cc, are identical except for their 8 or 22 amino acid C-termini. The PP1γ2 isoform is enriched in testis and is present in sperm from all placental mammals. Targeted disruption of Ppp1cc results in male infertility. Testis specific expression of PP1γ2 restores fertility in mice lacking Ppp1cc. However, replacing PP1γ2 with PP1γ1 in testis and sperm through CRISPR /Cas9 gene editing impairs sperm motility, ATP generation, and fertility. The interactions of protein phosphatase 1 (RIPPO) - PPP1R2/I2, PPP1R7/SDS22, and PPP1R3/I3 - are present in sperm as regulators of PP1γ2 during sperm motility initiation and maturation in the epididymis. Reversible phosphorylation of these RIPPOs results in PP1γ2 activation or inhibition. The spatiotemporal expression of I2, I3, and sds22 matches the expression of PP1γ2 in developing testicular spermatogenic cells. The enzyme PP1γ2 and the inhibitors colocalize within sperm. In PP1γ1 knock-in mice, I2, I3, and SDS22 localization within sperm are dramatically altered. While PP1γ2 and the RIPPOs are present in the functional regions of the sperm head, PP1γ1 along with I2, I3, and SDS22 are absent. Protein levels, of the inhibitors determined by mass spectroscopy and western blot are similar to that found in testis and sperm of WT mice. The mRNA levels of SDS22, I2, I3 are also roughly similar in testis KI mice. We have determined levels of phosphorylation of I2, I3, and SDS22 in sperm from PP1γ1 KI PP1γ2 mice compared to WT mice in caudal sperm. Serine phosphorylation of PPP1R7 and PPP1R2 were significantly altered in sperm from PP1γ1 knock-in mice while phosphorylation of PPP1R11 was unchanged, Phosphoproteome analysis also showed altered phosphorylation of hexokinase and mitochondrial pyruvate dehydrogenase. The phosphorylation of mono carboxylate transporter along with its associated protein embigin and spetin present in sperm midpiece and annulus are altered. Phosphorylation of Izumo which is present in sperm head and responsible sperm binding to the egg is altered. Metabolome determination showed lowering of the intermediates of the glycolytic pathway. The levels of ATP were also significantly lower. While the reasons for the altered localization of PP1γ1 and the RIPOs are still unknown, the altered intra-sperm phosphatase landscape is the likely basis for altered phosphorylation of these interactors and the other proteins involved in sperm metabolism and egg binding. This PP1γ1 knock-in mouse model is anticipated to enhance our understanding the reasons for specific requirement of PP1γ2 in sperm from mammals. Results from this and ongoing studies should provide basic and clinical insights into the basis for fertility and infertility in men.
Part II: Epididymal transit renders key competence to mammalian spermatozoa for fertilizing eggs. Generally, the two paralogs of glycogen synthase kinase 3, GSK3α and GSK3β, functionally overlap except in testis and sperm. We showed that GSK3α is essential for epididymal sperm maturation and fertilization. Male infertility is the only phenotype of mice with a global or testis-specific knockout (KO) of Gsk3α. Their sperm maturation is impaired, and sperm cannot fertilize eggs in vitro and in vivo. This suggests that GSK3α is a “male fertility kinase” in mammals and that GSK3α-selective inhibitor is a potential male contraceptive. A set of eight heterozygous Gsk3α (±) male mice received daily intraperitoneal injections of BRD0705, an isoform-selective GSK3α inhibitor, at 20 mg/kg body weight for 1 week. Five vehicle-treated and BRD0705-treated mice were tested for in vivo fertility and the remaining mice were sacrificed; their caudal spermatozoa were examined for motility and biochemical properties. The treated mice did not sire any pups while the control group sired 46 pups with a normal gestation period of 19–23 days. Continued fertility testing up to 6 weeks post-treatment, showed that the treated mice regained fertility siring 56 pups, with 76 in the control group. Sperm motility was impaired, its abnormal morphology increased during epididymal transit, Adenosine triphosphate (ATP) levels were low, and tyrosine- phosphorylation of hexokinase was absent: these phenotypes imitated those observed in Gsk3a KO mice. Tyrosine279-phosphorylation of GSK3α was reduced in sperm from the treated mice showing that the GSK3α activity was inhibited. The altered sperm phenotypes returned to normal following recovery of fertility. Complete infertility resulted after 1 week of BRD0705-treatment and fertility recovered after cessation of the treatment. Work is ongoing to determine the minimum dose and treatment time and the testing of new compounds with increased selectivity and inhibitory activity against GSK3α.
Description
Discussion I (A/B)
The serine/threonine phosphatase, PP1, has the paralogs - PP1α, PP1β, PP1γ1 – encoded by three genes. The enzyme PP1 encoded by Ppp1cc is expressed at two isoforms – PP1γ1 and PP1γ2 – by alternate splicing. In all cells and tissues, except testis, the paralogs are functionally interchangeable substituting for each other with the loss of one or more of the four forms. The only phenotype with the loss of Ppp1cc is male infertility due to the absence of PP1γ2 in testis. This isoform in expressed in testis and present in sperm from all mammals due to splicing of PPP1cc transcript. Thus, PP1γ2 is mammal and sperm specific. It is surprising that the other three isoforms, PP1α, PP1β, and PP1γ1 – are excluded from developing spermatogenic cells and sperm. Unlike PP1γ2, PP1γ1 cDNA could not be expressed as a transgene in testis presumably because of the presence of testis-expressed miRNA targets in the 3’ untranslated region of the PP1γ1 transcript. To understand the basis for the exclusive requirement of PP1γ2 a KI mouse line was generated by Crisper/Cas9 edited removal of the region which form the 3’ untranslated region of the PP1γ1 transcript and change the splice site to prevent splicing to form PP1γ2. This change in the gene is in essence results in an isoform specific KO of PP1γ2 while enabling PP1γ1 expression. This change in the gene enable robust expression of PP1γ1 in testis and its incorporation into sperm. The KI mice produced PP1 bearing sperm whereas WT mice produce PP1γ2 bearing sperm.
Sperm from PP1γ1 KI mice were infertile. The edited Ppp1cc gene directed expression of PP1γ1 at the same level as PP1γ2 in WT testis. Messenger RNA levels, protein levels and distribution within testis was indistinguishable from PP1γ2 in WT testis. Protein levels in sperm were also like PP1γ2 in WT sperm. The major difference was the striking absence of the PP1γ1 enzyme in the sperm head. In WT sperm PP1γ2 is present in the equatorial region of the sperm head, a region involved in sperm binding to the egg. Thus PP1γ1, at the same levels of PP1γ2 in WT, is now present in the flagellum of KI sperm. The regulators of PP1, I2, I3 and sds22, which in WT sperm have distinct localization along with PP1γ2 in the head, like PP1γ1 are also absent in the heads of sperm from KI mice. The inhibitors are now localized with PP1γ1 along the flagellum. Absence of the inhibitors in the sperm head is not due changes in their expression levels in testis and sperm. The protein levels of the inhibitors are same levels as in WT sperm. This drastic change in localization of the phosphatase and its regulators are likely to change activity in the sperm and head and tail.
While activity measurements were not possible because the enzyme was in the insoluble fraction of sperm homogenates there were changes in the phosphorylation of protein found in the sperm head and tail. Some of the proteins identified were metabolic enzymes, transporters and proteins involved in fertilization and motility. Loss of tyrosine phosphorylation of hexokinase, a rate controlling enzyme in glycolysis, was in seen and confirmed by MS and western blot analysis. Tyrosine phosphorylation of hexokinase regulates its activity. The block in hexokinase activity is also reflected in the low levels of the intermediates in the glycolytic pathway. Lowered ATP production was also confirmed in the metabolome analysis as seen in luciferase assay of sperm extracts. Phosphorylation changes in the bidirectional mono carboxylate transporter (MCT2) is also presumable responsible for lowered ATP with lactate and pyruvate. Changes in phosphorylation of Izumo, a protein responsible for sperm binding to the egg, is notable and possibly one of the reasons for lowered fertility of sperm. The function of the sperm enzyme cytoplasmic 5’ nucleotidase which undergoes several phosphorylation changes in KI sperm is not known. This enzyme has been implicated in regulating energy charge ratios in normal and cancer cells. Changes in the activity of the enzyme could underly changes in the adenosine nucleotide levels in the KI sperm. In our previous studies have shown that phosphorylation of SDS22 is altered in the absence of GSK3α. In KI mice sperm, we identified notably raised LFQ values of GSK3α phosphorylation, while those of GSK3β remained unchanged. This increase in phosphorylation could be attributed to the absence of its regulatory phosphatase PP1γ2 in the KI sperm head. Furthermore, we found a significant reduces in the phosphorylation of SPACA1 and IZUMO1, both essential for sperm binding to the egg and fertilization. Phospho-proteomics data also detected decreased phosphorylation levels of SPACA1, IZUMO1, I2, and SDS22, likely due to increased phosphorylation of GSK3α, as validated by Western blot analysis. Our findings suggest that PP1γ1 failed to interact with GSK3α, resulting in its sustained phosphorylation. Notably, SPACA1, containing a GSK3-consensus motif near its C-terminus, undergoes cleavage, leading to the localization shift of IZUMO1 from the post-acrosomal to the equatorial region for sperm-ovum fertilization [88]. The raised phosphorylation of GSK3α then reduced its activity, associated with decreased phosphorylation of SPACA1, IZUMO1, I2, and SDS22, as indicated by the phosphor-proteome.
In sum the infertility, motility and energy level impairment is a result of the changes in the phosphorylation of the proteins involved the key question why there is such a striking altered intra-sperm localization PP1γ1 is a mystery. The answer to this question will also shed light on why PP1γ2 is uniquely required and irreplaceable only in mammalian sperm. There may be a scaffolding protein that directs the enzyme to the equatorial region of the sperm head may be missing in KI sperm. It is possible that the expression of this currently unknown protein may be expressed in testis in isoform specific manner by PP1γ2. There is evidence that the transcription factor Spz1, which is testis and mammal specific, binds preferentially to PP1γ2. Further studies should unravel the identity of the proteins expressed under the isoform specific gene expression function of PP1γ2. These studies using proteomic determination and identification genes expressed under the regulation of the transcription factor Spz1 are in progress. Despite this uncertainty results from this KI mouse has shed light on how changes in metabolic enzymes and protein involved in sperm binding to egg and flagellar proteins controlling motility affect male fertility. This information likely has both basic and clinical significance.
The enzyme GSK3α isoform is essential and irreplaceable for sperm fertility in mice. Male infertility is the only phenotype seen in mice with a global loss of GSK3α. This isoform is conserved and present only in sperm from placental mammals [81]: species where epididymal sperm maturation and pre-fertilization events in the female reproductive are unique requirements for male fertility. These findings together formed a strong basis to explore the possibility of targeting GSK3α in sperm during their residence in the epididymis. Exploiting a single amino acid difference in the ATP binding domain of GSK3, Wagner, and co-workers was able to develop paralog selective inhibitors for GSK3.33 Of these, the compound BRD0705 had approximately 8-fold selective inhibition for GSK3α with little off- target effects on other protein kinases. There were no observable adverse effects in the in vivo studies. We used the same drug dose as used in the AML studies by Wagner et al. for targeting GSK3α in mice. [84] We chose GSK3a(±) mice for these studies. We had previously shown that sperm from the GSK3a (±) mice expressed 75% of GSK3α com- pared to sperm from wild-type mice. [80] Thus, the heterozygous mice were the choice for these initial exploratory studies. Male mice treated for 1 week with BRD0705 were rendered infertile. Mating was normal as seen in the examination of vaginal plugs. Following the cessation of treatment, fertility was almost completely restored in the treated mice. The phenotype of the treated mice was similar to that seen in Gsk3α KO mice. That is, the outcome of the pharmacologic GSK3α inhibition phenocopies the genetic loss of Gsk3α further emphasizing the role of an active GSK3α during epididymal sperm maturation. A decrease in motility and an increase in abnormal morphologies were also observed. The percentage of morphological abnormality in spermatozoa from caput to cauda significantly increased (Figure 37C, D), whereas it was non-significant in testicular sperm. This indicates serious maturational defects as a result of BRD0705 doses, without nullifying its adverse effect in spermatogenesis, if any. The junction of sperm midpiece- principal piece was highly affected by the inhibition of GSK3α in vivo. An impaired tail structure greatly affects sperm progressive as well as hyperactive movement, justifying the selection of this inhibitor for a contraceptive preparation.
Phosphorylation of hexokinase returned in sperm from mice with restored fertility. Reduced GSK3α tyrosine279 phosphorylation, an indicator of enzyme activity inhibition, was seen in sperm from drug- treated mice. Tyrosine phosphorylation of GSK3α also recovered to control levels along with restoration of fertility following cessation of the drug treatment. The finding that β-catenin levels remained unchanged, along with normal sperm counts and testis histology, suggests that GSK3β paralogs in the testis were unaffected by the drug treatment. This significantly enhances the utility of BRD0705, as it guarantees that no adverse effects will arise from β-catenin stabilization associated with this specific inhibitor. While our data clearly show that GSK3α in sperm was affected, whether GSK3α in other tissues was affected was not determined. Our data also indicate that the treatment protocol resulted in sufficient bioavailability of BRD0705 in the epididymis to inhibit sperm GSK3α leading to infertility. Sperm from drug-treated mice were similar to sperm from Gsk3α KO mice. Work is in progress to determine minimum drug levels and treatment time and the testing of new compounds with increased selectivity and inhibitory activity against GSK3α.
Keywords
ISOFORM-SPECIFIC ROLE OF PP1𝛾 2 IN SPERMFUNCTION AND MALE FERTILITY KINASE IN MAMMALS, A POTEINTAIL NON-HORMONAL CONTRACEPTIVE TARGETING EPIDIDYMAL SPERM
Citation
03 January 2025 https://doi.org/10.1111/andr.13838 and Reproduction. 2025 February 01; 169(2): . doi:10.1530/REP-24-0256.