Panobinostat enhances olaparib efficacy by modifying expression of homologous recombination repair and immune transcripts in ovarian cancer.

Histone deacetylase inhibitors (HDACi) sensitize homologous recombination (HR)-proficient human ovarian cancer cells to PARP inhibitors (PARPi). To investigate mechanisms of anti-tumor effects of combined HDACi/PARPi treatment we performed transcriptome analysis in HR- proficient human ovarian cancer cells and tested drug effects in established immunocompetent mouse ovarian cancer models. Human SKOV-3 cells were treated with vehicle (Con), olaparib (Ola), panobinostat (Pano) or Pano+Ola and RNA-seq analysis performed. DESeq2 identified differentially expressed HR repair and immune transcripts. Luciferised syngeneic mouse ovarian cancer cells (ID8-luc) were treated with the HDACi panobinostat alone or combined with olaparib and effects on cell viability, apoptosis, DNA damage and HR efficiency determined. C57BL/6 mice with intraperitoneally injected ID8-luc cells were treated with panobinostat and/or olaparib followed by assessment of tumor burden, markers of cell proliferation, apoptosis and DNA damage, tumor-infiltrating T cells and macrophages, and other immune cell populations in ascites fluid. There was a significant reduction in expression of 20/37 HR pathway genes by Pano+Ola, with immune and inflammatory-related pathways also significantly enriched by the combination. In ID8 cells, Pano+Ola decreased cell viability, HR repair, and enhanced DNA damage. Pano+Ola also co-operatively reduced tumor burden and proliferation, increased tumor apoptosis and DNA damage, enhanced infiltration of CD8+ T cells into tumors, and decreased expression of M2-like macrophage markers. In conclusion, panobinostat in combination with olaparib targets ovarian tumors through both direct cytotoxic and indirect immune-modulating effects.

Entinostat, a selective HDAC1/2 inhibitor, potentiates the effects of olaparib in homologous recombination proficient ovarian cancer.

OBJECTIVE: Poly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer. METHODS: We assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model. RESULTS: Entinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability. CONCLUSION: Entinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.

Ascites-induced compression alters the peritoneal microenvironment and promotes metastatic success in ovarian cancer.

The majority of women with recurrent ovarian cancer (OvCa) develop malignant ascites with volumes that can reach > 2 L. The resulting elevation in intraperitoneal pressure (IPP), from normal values of 5 mmHg to as high as 22 mmHg, causes striking changes in the loading environment in the peritoneal cavity. The effect of ascites-induced changes in IPP on OvCa progression is largely unknown. Herein we model the functional consequences of ascites-induced compression on ovarian tumor cells and components of the peritoneal microenvironment using a panel of in vitro, ex vivo and in vivo assays. Results show that OvCa cell adhesion to the peritoneum was increased under compression. Moreover, compressive loads stimulated remodeling of peritoneal mesothelial cell surface ultrastructure via induction of tunneling nanotubes (TNT). TNT-mediated interaction between peritoneal mesothelial cells and OvCa cells was enhanced under compression and was accompanied by transport of mitochondria from mesothelial cells to OvCa cells. Additionally, peritoneal collagen fibers adopted a more linear anisotropic alignment under compression, a collagen signature commonly correlated with enhanced invasion in solid tumors. Collectively, these findings elucidate a new role for ascites-induced compression in promoting metastatic OvCa progression.

CYR61/CCN1 Regulates dCK and CTGF and Causes Gemcitabine-resistant Phenotype in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) develops extrinsic- and intrinsic-resistant phenotypes to prevent chemotherapies from entering into the cells by promoting desmoplastic reactions (DR) and metabolic malfunctions of the drugs. It is well established that these responses are also associated with pancreatic cancer cells' gemcitabine resistance. However, the mechanism by which these resistant pathways function in the pancreatic cancer cells remains poorly understood. In these studies, we show that CYR61/CCN1 signaling plays a vital role in making pancreatic cancer cells resistant to gemcitabine in vitro and also in a tumor xenograft model. We proved that the catastrophic effect of gemcitabine could significantly be increased in gemcitabine-resistant PDAC cells when CYR61/CCN1 is depleted, while this effect can be suppressed in gemcitabine-sensitive neoplastic cells by treating them with CYR61/CCN1 recombinant protein. Ironically, nontransformed pancreatic cells, which are sensitive to gemcitabine, cannot be resistant to gemcitabine by CYR61/CCN1 protein treatment, showing a unique feature of CYR61/CCN signaling that only influences PDAC cells to become resistant. Furthermore, we demonstrated that CYR61/CCN1 suppresses the expression of the gemcitabine-activating enzyme deoxycytidine kinase (dCK) while it induces the expression of a DR-promoting factor CTGF (connective tissue growth factor) in pancreatic cancer cells in vitro and in vivo Thus, the previously described mechanisms (dCK and CTGF pathways) for gemcitabine resistance may be two novel targets for CYR61/CCN1 to protect pancreatic cancer cells from gemcitabine. Collectively, these studies reveal a novel paradigm in which CYR61/CCN1regulates both extrinsic and intrinsic gemcitabine resistance in PDAC cells by employing unique signaling pathways.

N-Butyldeoxygalactonojirimycin Induces Reversible Infertility in Male CD Rats.

This study shows for the first time that an iminosugar exerts anti-spermiogenic effect, inducing reversible infertility in a species that is not related to C57BL/6 male mice. In CD rats, N-butyldeoxygalactonojirimycin (NB-DGJ) caused reversible infertility at 150 mg/kg/day when administered daily as single oral dose. NB-DGJ inhibited CD rat-derived testicular ß-glucosidase 2 (GBA2) activity at 10 µM but did not inhibit CD rat-derived testicular ceramide-specific glucosyltransferase (CGT) at doses up to 1000 µM. Pharmacokinetic studies revealed that sufficient plasma levels of NB-DGJ (50 µM) were achieved to inhibit the enzyme. Fertility was blocked after 35 days of treatment and reversed one week after termination of treatment. The rapid return of fertility indicates that the major effect of NB-DGJ may be epididymal rather than testicular. Collectively, our in vitro and in vivo studies in rats suggest that iminosugars should continue to be pursued as potential lead compounds for development of oral, non-hormonal male contraceptives. The study also adds evidence that GBA2, and not CGT, is the major target for the contraceptive effect of iminosugars.

Protein PEGylation for cancer therapy: bench to bedside.

PEGylation is a biochemical modification process of bioactive molecules with polyethylene glycol (PEG), which lends several desirable properties to proteins/peptides, antibodies, and vesicles considered to be used for therapy or genetic modification of cells. However, PEGylation of proteins is a complex process and can be carried out using more than one strategy that depends on the nature of the protein and the desired application. Proteins of interest are covalently conjugated or non-covalently complexed with inert PEG strings. Purification of PEGylated protein is another critical step, which is mainly carried out based on electrostatic interactions or molecular sizes using chromatography. Several PEGylated drugs are being used for diseases like anemia, kidney disease, multiple sclerosis, hemophilia and cancers. With the advancement and increased specificity of the PEGylation process, the world of drug therapy, and specifically cancer therapy could benefit by utilizing this technique to create more stable and non-immunogenic therapies. In this article we describe the structure and functions of PEGylation and how this chemistry helps in drug discovery. Moreover, special emphasis has been given to CCN-family proteins that can be targeted or used as therapy to prevent or block cancer progression through PEGylation technology.

Bipolar Tumor-Associated Macrophages in Ovarian Cancer as Targets for Therapy.

Ovarian cancer, a rare but fatal disease, has been a challenging area in the field of gynecological cancer. Ovarian cancer is characterized by peritoneal metastasis, which is facilitated by a cross-talk between tumor cells and other cells in the tumor microenvironment (TME). In epithelial ovarian cancer, tumor-associated macrophages (TAMs) constitute over 50% of cells in the peritoneal TME and malignant ascites, and are potential targets for therapy. Here, we review the bipolar nature of TAMs and the evolving strategies to target TAMs in ovarian cancer.

The MAZ transcription factor is a downstream target of the oncoprotein Cyr61/CCN1 and promotes pancreatic cancer cell invasion via CRAF-ERK signaling.

Myc-associated zinc-finger protein (MAZ) is a transcription factor with dual roles in transcription initiation and termination. Deregulation of MAZ expression is associated with the progression of pancreatic ductal adenocarcinoma (PDAC). However, the mechanism of action of MAZ in PDAC progression is largely unknown. Here, we present evidence that MAZ mRNA expression and protein levels are increased in human PDAC cell lines, tissue samples, a subcutaneous tumor xenograft in a nude mouse model, and spontaneous cancer in the genetically engineered PDAC mouse model. We also found that MAZ is predominantly expressed in pancreatic cancer stem cells. Functional analysis indicated that MAZ depletion in PDAC cells inhibits invasive phenotypes such as the epithelial-to-mesenchymal transition, migration, invasion, and the sphere-forming ability of PDAC cells. Mechanistically, we detected no direct effects of MAZ on the expression of K-Ras mutants, but MAZ increased the activity of CRAF-ERK signaling, a downstream signaling target of K-Ras. The MAZ-induced activation of CRAF-ERK signaling was mediated via p21-activated protein kinase (PAK) and protein kinase B (AKT/PKB) signaling cascades and promoted PDAC cell invasiveness. Moreover, we found that the matricellular oncoprotein cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) regulates MAZ expression via Notch-1-sonic hedgehog signaling in PDAC cells. We propose that Cyr61/CCN1-induced expression of MAZ promotes invasive phenotypes of PDAC cells not through direct K-Ras activation but instead through the activation of CRAF-ERK signaling. Collectively, these results highlight key molecular players in PDAC invasiveness and may help inform therapeutic strategies to improve clinical management and outcomes of PDAC.

Racial disparity in breast cancer: can it be mattered for prognosis and therapy.

Breast cancer (BC) has emerged as a deadly disease that affects the lives of millions of women worldwide. It is the second leading cause of cancer-related deaths in the United States. Advancements in BC screening, preventive measures and treatment have resulted in significant decline in BC related deaths. However, unacceptable levels of racial disparity have been consistently reported, especially in African-American (AA) women compared to European American (EA). AA women go through worse prognosis, shorter survival time and higher mortality rates, despite higher cancer incidence reported in EA. These disparities are independent of socioeconomic status, access to healthcare or age, or even the stage of BC. Recent race-specific genetic and epigenetic studies have reported biological causes, which form the crux of this review. However, the developments are just the tip of the iceberg. Prioritizing primary research towards studying race-specific tumor microenvironment and biological composition of the host system in delineating the cause of these disparities is utmost necessary to ameliorate the disparity and design appropriate diagnosis/treatment regimen for AA women suffering from BC. In this review article, we discuss emerging trends and exciting discoveries that reveal how genetic/epigenetic circuitry contributed to racial disparity and discussed the strategies that may help in future therapeutic development.

Structure-Activity Studies of N-Butyl-1-deoxynojirimycin (NB-DNJ) Analogues: Discovery of Potent and Selective Aminocyclopentitol Inhibitors of GBA1 and GBA2.

Analogues of N-butyl-1-deoxynojirimycin (NB-DNJ) were prepared and assayed for inhibition of ceramide-specific glucosyltransferase (CGT), non-lysosomal ß-glucosidase 2 (GBA2) and the lysosomal ß-glucosidase 1 (GBA1). Compounds 5 a-6 f, which carry sterically demanding nitrogen substituents, and compound 13, devoid of the C3 and C5 hydroxy groups present in DNJ/NB-DGJ (N-butyldeoxygalactojirimycin) showed no inhibitory activity for CGT or GBA2. Inversion of stereochemistry at C4 of N-(n-butyl)- and N-(n-nonyl)-DGJ (compounds 24) also led to a loss of activity in these assays. The aminocyclopentitols N-(n-butyl)- (35 a), N-(n-nonyl)-4-amino-5-(hydroxymethyl)cyclopentane- (35 b), and N-(1-(pentyloxy)methyl)adamantan-1-yl)-1,2,3-triol (35 f), were found to be selective inhibitors of GBA1 and GBA2 that did not inhibit CGT (>1 mm), with the exception of 35 f, which inhibited CGT with an IC50 value of 1 mm. The N-butyl analogue 35 a was 100-fold selective for inhibiting GBA1 over GBA2 (Ki values of 32 nm and 3.3 µm for GBA1 and GBA2, respectively). The N-nonyl analogue 35 b displayed a Ki value of ≪14 nm for GBA1 inhibition and a Ki of 43 nm for GBA2. The N-(1-(pentyloxy)methyl)adamantan-1-yl) derivative 35 f had Ki values of ≈16 and 14 nm for GBA1 and GBA2, respectively. The related N-bis-substituted aminocyclopentitols were found to be significantly less potent inhibitors than their mono-substituted analogues. The aminocyclopentitol scaffold should hold promise for further inhibitor development.

Human pancreatic cancer progression: an anarchy among CCN-siblings.

Decades of basic and translational studies have identified the mechanisms by which pancreatic cancer cells use molecular pathways to hijack the normal homeostasis of the pancreas, promoting pancreatic cancer initiation, progression, and metastasis, as well as drug resistance. These molecular pathways were explored to develop targeted therapies to prevent or cure this fatal disease. Regrettably, the studies found that majority of the molecular events that dictate carcinogenic growth in the pancreas are non-actionable (potential non-responder groups of targeted therapy). In this review we discuss exciting discoveries on CCN-siblings that reveal how CCN-family members contribute to the different aspects of the development of pancreatic cancer with special emphasis on therapy.

Exosomes in carcinogenesis: molecular palkis carry signals for the regulation of cancer progression and metastasis.

Exosomes, which act as biological cargo vessels, are cell-released, phospholipid-enclosed vesicles. In eukaryotic cells, exosomes carry and exchange biological materials or signals for the benefit or detriment to the cells. Thereby, we consider exosomes to be molecular Palkis (carriers). Although exosomes are currently one of the most popularly researched cellular entities, they have remained largely enigmatic and warrant continued investigation into their structure and functions. These membraned vesicles are between 30 and 150 nm in diameter and are actively secreted by all cell types. While initially considered cellular "trash bags," recent years have revealed exosomes to be dynamic and multi-functional vesicles that may play a crucial role in cancer development, progression and metastasis. Thereby, they have the potential to be used in development of therapeutic modalities for cancer and other diseases. As more research studies emerge, it's becoming evident that exosomes are released by cells with a purpose and are representatives of certain cell types and disease conditions. Hence, they may also be used as biomarkers for the detection of cancer initiation, progression and organotropic metastatic growth of cancer cells. This review will focus on the recent developments achieved in identifying the role of exosomes in cancer development and progression as well as therapeutic implications. The review will also discuss the pitfalls of methodologies used for the extraction of exosomes.

Effect of Cocaine on Pulmonary Vascular Remodeling and Hemodynamics in Human Immunodeficiency Virus-Transgenic Rats.

Human immunodeficiency virus (HIV)-related pulmonary arterial hypertension has been found to be more prevalent in intravenous drug users. Our earlier cell-culture findings reported down-regulation of bone morphogenetic protein receptors (BMPRs) in combination with enhanced proliferation of human pulmonary arterial smooth muscle cells (PASMCs) in the presence of HIV-Trans-activator of transcription (Tat) and cocaine compared with either treatment alone. Here, we report physiologic evidence of significant increases in mean pulmonary arterial pressure in HIV-transgenic (Tg) rats intraperitoneally administered 40 mg/kg body weight cocaine (HIV-cocaine group) once daily for 21 days when compared with HIV-Tg rats given saline (HIV group) or wild-type (WT) Fischer 334 rats treated with (WT-cocaine group) and without cocaine (WT group). In addition, right ventricle systolic pressure was also found to be significantly higher in the HIV-cocaine rats compared with the WT group. Significant down-regulation in protein expression of BMPR-2 and BMPR-1B was observed in total lung extract from HIV-cocaine rats compared with the other three groups. Furthermore, the PASMCs isolated from HIV-cocaine rats demonstrated a higher level of proliferation and lower levels of apoptosis compared with cells isolated from other rat groups. Interestingly, corroborating our earlier cell-culture findings, we observed higher expression of BMPR-2 and BMPR-1B messenger RNA and significantly lower levels of BMPR-2 and BMPR-1B protein in HIV-cocaine PASMCs compared with cells isolated from all other groups. In conclusion, our findings support an additive effect of cocaine and HIV on smooth muscle dysfunction, resulting in enhanced pulmonary vascular remodeling with associated elevation of mean pulmonary arterial pressure and right ventricle systolic pressure in HIV-Tg rats exposed to cocaine.

MicroRNA Expression Signatures During Malignant Progression From Barrett's Esophagus.

The rapid increase and poor survival of esophageal adenocarcinoma (EAC) have led to significant efforts to promote early detection. Given that the premalignant lesion of Barrett's esophagus (BE) is the major known risk factor for EAC, multiple investigators have studied biomarker signatures that can predict malignant progression of BE to EAC. MicroRNAs, a novel class of gene regulators, are small non-coding RNAs and have been associated with carcinogenesis. MicroRNAs are ideal biomarkers because of their remarkable stability in fixed tissues, a common method for collection of clinical specimens, and in blood either within exosomes or as microRNA-protein complexes. Multiple studies show potential of microRNAs as tissue and blood biomarkers for diagnosis and prognosis of EAC but the results need confirmation in prospective studies. Although head-to-head comparisons are lacking, microRNA panels require less genes than messenger RNA panels for diagnosis of EAC in BE. MicroRNA diagnostic panels will need to be compared for accuracy against global measures of genome instability that were recently shown to be good predictors of progression but require sophisticated analytic techniques. Early studies on blood microRNA panels are promising but have found microRNA markers to be inconsistent among studies. MicroRNA expression in blood is different between various microRNA sub-compartments such as exosomes and microRNA-protein complexes and could affect blood microRNA measurements. Further standardization is needed to yield consistent results. We have summarized the current understanding of the tissue and blood microRNA signatures that may predict the development and progression of EAC.

A tissue retrieval and postharvest processing regimen for rodent reproductive tissues compatible with long-term storage on the international space station and postflight biospecimen sharing program.

Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at -80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

Ligand-Independent Activation of Platelet-Derived Growth Factor Receptor ß during Human Immunodeficiency Virus-Transactivator of Transcription and Cocaine-Mediated Smooth Muscle Hyperplasia.

Our previous study supports an additive effect of cocaine to human immunodeficiency virus infection in the development of pulmonary arteriopathy through enhancement of proliferation of pulmonary smooth muscle cells (SMCs), while also suggesting involvement of platelet-derived growth factor receptor (PDGFR) activation in the absence of further increase in PDGF-BB ligand. Redox-related signaling pathways have been shown to regulate tyrosine kinase receptors independent of ligand binding, so we hypothesized that simultaneous treatment of SMCs with transactivator of transcription (Tat) and cocaine may be able to indirectly activate PDGFR through modulation of reactive oxygen species (ROS) without the need for PDGF binding. We found that blocking the binding of ligand using suramin or monoclonal IMC-3G3 antibody significantly reduced ligand-induced autophosphorylation of Y1009 without affecting ligand-independent transphosphorylation of Y934 residue on PDGFRß in human pulmonary arterial SMCs treated with both cocaine and Tat. Combined treatment of human pulmonary arterial SMCs with cocaine and Tat resulted in augmented production of superoxide radicals and hydrogen peroxide when compared with either treatment alone. Inhibition of this ROS generation prevented cocaine- and Tat-mediated Src activation and transphosphorylation of PDGFRß at Y934 without any changes in phosphorylation of Y1009, in addition to attenuation of smooth muscle hyperplasia. Furthermore, pretreatment with an Src inhibitor, PP2, also suppressed cocaine- and Tat-mediated enhanced Y934 phosphorylation and smooth muscle proliferation. Finally, we report total abrogation of cocaine- and Tat-mediated synergistic increase in cell proliferation on inhibition of both ligand-dependent and ROS/Src-mediated ligand-independent phosphorylation of PDGFRß.

Synthesis and evaluation of eight- and four-membered iminosugar analogues as inhibitors of testicular ceramide-specific glucosyltransferase, testicular ß-glucosidase 2, and other glycosidases.

Eight- and four-membered analogues of N-butyldeoxynojirimycin (NB-DNJ), a reversible male contraceptive in mice, were prepared and tested. A chiral pool approach was used for the synthesis of the target compounds. Key steps for the synthesis of the eight-membered analogues involve ring-closing metathesis and Sharpless asymmetric dihydroxylation and for the four-membered analogues Sharpless epoxidation, epoxide ring-opening (azide), and Mitsunobu reaction to form the four-membered ring. (3S,4R,5S,6R,7R)-1-Nonylazocane-3,4,5,6,7-pentaol (6) was moderately active against rat-derived ceramide-specific glucosyltransferase, and four of the other eight-membered analogues were weakly active against rat-derived ß-glucosidase 2. Among the four-membered analogues, ((2R,3S,4S)-3-hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (25) displayed selective inhibitory activity against mouse-derived ceramide-specific glucosyltransferase and was about half as potent as NB-DNJ against the rat-derived enzyme. ((2S,4S)-3-Hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (27) was found to be a selective inhibitor of ß-glucosidase 2, with potency similar to NB-DNJ. Additional glycosidase assays were performed to identify potential other therapeutic applications. The eight-membered iminosugars exhibited specificity for almond-derived ß-glucosidase, and the 1-nonylazetidine 25 inhibited α-glucosidase (Saccharomyces cerevisiae) with an IC(50) of 600 nM and ß-glucosidase (almond) with an IC(50) of 20 µM. Only N-nonyl derivatives were active, emphasizing the importance of a long lipophilic side chain for inhibitory activity of the analogues studied.

Role of FYN kinase in spermatogenesis: defects characteristic of Fyn-null sperm in mice.

FYN kinase is highly expressed in the testis and has been implicated in testis and sperm function, yet specific roles for this kinase in testis somatic and germ cells have not been defined. The purpose of the present investigation was to identify aspects of spermatogenesis, spermiation, or sperm fertilizing capacity that required FYN for normal reproductive function. Matings between Fyn-null males and wild-type females resulted in normal litter sizes, despite the fact that Fyn-null males exhibited reduced epididymal size and sperm count. Morphological analysis revealed a high frequency of abnormal sperm morphology among Fyn-null sperm, and artificial insemination competition studies demonstrated that Fyn-null sperm possessed reduced fertilizing capacity. Fyn-null sperm exhibited nearly normal motility during capacitation in vitro but reduced ability to undergo the acrosome reaction and fertilize oocytes. The typical pattern of capacitation-induced protein tyrosine phosphorylation was slightly modified in Fyn-null sperm, with reduced abundance of several minor phosphoproteins. These findings are consistent with a model in which FYN kinase plays an important role in proper shaping of the head and acrosome within the testis and possibly an additional role in the sperm acrosome reaction, events required for development of full fertilizing capacity in sperm.