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Mustfa Thesis (Dissertation).pdf (46.33 MB)

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ISOFORM-SPECIFIC ROLE OF PP1γ 2 IN SPERMFUNCTION AND MALE FERTILITY KINASE IN MAMMALS, A POTEINTAIL NON-HORMONAL CONTRACEPTIVE TARGETING EPIDIDYMAL SPERM

Kabi, Mustfa
http://rave.ohiolink.edu/etdc/view?acc_num=kent1740821854714954

Abstract Details

2025, PHD, Kent State University, College of Arts and Sciences / School of Biomedical Sciences.
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 interactors 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 Gsk3α 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.
Srinivasan Vijayaraghavan, Dr (Advisor)
139 p.
Biology; Biomedical Research

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Citations

  • Kabi, M. (2025). ISOFORM-SPECIFIC ROLE OF PP1γ 2 IN SPERMFUNCTION AND MALE FERTILITY KINASE IN MAMMALS, A POTEINTAIL NON-HORMONAL CONTRACEPTIVE TARGETING EPIDIDYMAL SPERM [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1740821854714954

    APA Style (7th edition)

  • Kabi, Mustfa. ISOFORM-SPECIFIC ROLE OF PP1γ 2 IN SPERMFUNCTION AND MALE FERTILITY KINASE IN MAMMALS, A POTEINTAIL NON-HORMONAL CONTRACEPTIVE TARGETING EPIDIDYMAL SPERM. 2025. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1740821854714954.

    MLA Style (8th edition)

  • Kabi, Mustfa. "ISOFORM-SPECIFIC ROLE OF PP1γ 2 IN SPERMFUNCTION AND MALE FERTILITY KINASE IN MAMMALS, A POTEINTAIL NON-HORMONAL CONTRACEPTIVE TARGETING EPIDIDYMAL SPERM." Doctoral dissertation, Kent State University, 2025. http://rave.ohiolink.edu/etdc/view?acc_num=kent1740821854714954

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Kent State University and OhioLINK.