Biomedical subjects

David L Garbers

Publications and source records attributed to David L Garbers.

14 recordsLinked to original sources

PubMed2004

Defining the spermatogonial stem cell.

Through the use of donor cells from transgenic rats expressing GFP exclusively in the germline, we have defined culture conditions where male germ cells lose (on STO cells) or maintain (on MSC-1 cells) stem cell activity. A cadre of germ cell transcripts strikingly decrease in relative abundance as a function of testis age or culture time on STO cells, but only a subset of these transcripts (approximately 248) remain elevated when cultured on MSC-1 cells. If specific gene expression regulates stem cell activity, some or all of these transcripts are candidates as such regulators. We establish a spermatogonial stem cell index (SSCI) that reliably predicts relative stem cell activity in rat or mouse testis cell cultures, and through the use of an antibody to a robust signal (Egr3) within the index find intense signals in single or paired cells. As germ cells form longer interconnected chains (incomplete cytokinesis), the Egr3 signal disappears coincident with a loss of stem cell activity. Thus, molecular markers specific for spermatogonial stem cells establish a reliable and rapid means by which to define these cells in culture and alleviate the need for laborious testicular transfers in initial cell culture studies.

Animals↗

PubMed2004

A novel aminophospholipid transporter exclusively expressed in spermatozoa is required for membrane lipid asymmetry and normal fertilization.

Through the use of a functionally unbiased signal peptide trap screen, we have discovered an ATP-dependent aminophospholipid transporter that is exclusively expressed in the acrosomal region of spermatozoa; it is about 62% similar to the flippase, FIC1. We disrupted the transporter gene and found that the size of litters from male null mice was slightly smaller than found with wild-type males. Sperm morphology and motility were the same between null and wild-type littermates, but agents (merocyanine and annexin) that measure phospholipid packing or phosphatidylserine (PS) in the outer membrane leaflet showed that PS already existed in the outer leaflet of null spermatozoa before sperm capacitation. Fertilization rates were normal when null spermatozoa were added to zona pellucida-free eggs, but in the presence of the extracellular matrix, fewer transporter(-/-) spermatozoa bound tightly or penetrated the zona pellucida (ZP), and fewer underwent acrosome reactions. In vitro fertilization was compromised, especially at early time points or at low sperm concentrations after mixing null spermatozoa and eggs. Thus, a new aminophospholipid transporter expressed exclusively in spermatozoa is critical for normal phospholipid distribution in the bilayer, and for normal binding, penetration, and signaling by the zona pellucida.

Amino Acid Sequence↗

PubMed2003

Hyperactivated sperm motility driven by CatSper2 is required for fertilization.

Elevations of sperm Ca2+ seem to be responsible for an asymmetric form of motility called hyperactivation, which is first seen near the time of fertilization. The mechanism by which intracellular Ca2+ concentrations increase remains unknown despite considerable investigation. Although several prototypical voltage-gated calcium channels are present in spermatozoa, they are not essential for motility. Furthermore, the forward velocity and percentage of motility of spermatozoa are associated with infertility, but their importance relative to hyperactivation also remains unknown. We show here that disruption of the gene for a recently described sperm-specific voltage-gated cation channel, CatSper2, fails to significantly alter sperm production, protein tyrosine phosphorylation that is associated with capacitation, induction of the acrosome reaction, forward velocity, or percentage of motility, yet CatSper2-/- males are completely infertile. The defect that we identify in the null sperm cells is a failure to acquire hyperactivated motility, which seems to render spermatozoa incapable of generating the "power" needed for penetration of the extracellular matrix of the egg. A loss of power is suggested also by experiments in which the viscosity of the medium was increased after incubation of spermatozoa in normal capacitating conditions. In high-viscosity medium, CatSper2-null spermatozoa lost the ability to swim forward, whereas wild-type cells continued to move forward. Thus, CatSper2 is responsible for driving hyperactivated motility, and, even with typical sperm forward velocities, fertilization is not possible in the absence of this highly active form of motility.

Animals↗

PubMed2003

CatSper1 required for evoked Ca2+ entry and control of flagellar function in sperm.

CatSper family proteins are putative ion channels expressed exclusively in membranes of the sperm flagellum and required for male fertility. Here, we show that mouse CatSper1 is essential for depolarization-evoked Ca2+ entry and for hyperactivated movement, a key flagellar function. CatSper1 is not needed for other developmental landmarks, including regional distributions of CaV1.2, CaV2.2, and CaV2.3 ion channel proteins, the cAMP-mediated activation of motility by HCO3-, and the protein phosphorylation cascade of sperm capacitation. We propose that CatSper1 functions as a voltage-gated Ca2+ channel that controls Ca2+ entry to mediate the hyperactivated motility needed late in the preparation of sperm for fertilization.

Animals↗

PubMed2003

A new sperm-specific Na+/H+ exchanger required for sperm motility and fertility.

It has long been speculated that intracellular pH is a critical regulator of both invertebrate and vertebrate sperm motility, and sodium-hydrogen exchange has been suggested as a mediator of such pH(i) regulation in various instances. Two sodium-hydrogen exchangers (NHE1 and NHE5) are expressed in spermatozoa. However, elimination of the NHE1 gene fails to cause infertility, suggesting that normal sperm function is maintained in NHE1-null animals. Here, we used a functionally unbiased signal peptide trap screen to identify a novel sperm-specific NHE. The NHE contains 14 predicted transmembrane segments, including a potential voltage sensor and a consensus cyclic nucleotide-binding motif. Testis histology, sperm numbers and morphology were normal, but NHE-null males were completely infertile with severely diminished sperm motility. The addition of ammonium chloride, which elevates intracellular pH, partially rescued the motility and fertility defects. Surprisingly, cyclic AMP analogues almost completely rescued the motility and infertility phenotypes. The existence of this new sperm NHE provides an attractive contraceptive target, given its cell-specific expression and absolute requirement for fertility.

Ammonium Chloride↗

PubMed2003

Identification of a potent serum factor that causes desensitization of the receptor for C-Type natriuretic peptide.

BACKGROUND: Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum. Given the potential importance of this receptor in remodeling after tissue injury, identification of the serum factor(s) is of significant medical importance. RESULTS: Partial purification of desensitization activity in serum by DEAE-Sepharose and reverse phase C18 chromatography, followed by mass spectroscopy, identified peptide sequences identical to those of apolipoprotein A2 (Apo A2), a known component of high density lipoprotein (HDL). Apo A2, however, could be eliminated as the active desensitization factor. Never the less, substantial desensitization activity was associated with purified preparations of bovine or human HDL. Since HDL is a well-known transporter of various lipids and phospholipids, we extracted either HDL or partially purified serum preparations with butanol and all activity extracted into the solvent. Of various lipophilic signaling molecules known to be associated with HDL, a prominent component is sphingosine-1-phosphate (S1P). We therefore tested authentic S1P as well as other known components of HDL (sphingosylphosphorylcholine; platelet activating factor) for activity; only S1P caused desensitization of GC-B. S1P was relatively potent, causing one-half maximal desensitization of GC-B at concentrations of 5-10 nM. These effects were seen within a few minutes after addition. Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations. The pathway by which serum or S1P desensitizes GC-B seems unique in that pertussis toxin failed to inhibit GC-B desensitization, and yet blocked serum or S1P activation of extracellular signal-regulated kinase (ERK) or Akt/protein kinase B (Akt/PKB). CONCLUSION: Since the concentrations of S1P that desensitize GC-B are well within serum physiological ranges, this mitogenic signaling molecule likely functions as a strong adversary of the CNP/GC-B signaling pathway in the regulation of cell proliferation and other growth factor-induced phenotypes. The mechanism by which S1P desensitizes GC-B appears different than the known S1P signaling pathways.

Journal Article↗

PubMed2003

Androgen contributes to gender-related cardiac hypertrophy and fibrosis in mice lacking the gene encoding guanylyl cyclase-A.

Myocardial hypertrophy and extended cardiac fibrosis are independent risk factors for congestive heart failure and sudden cardiac death. Before age 50, men are at greater risk for cardiovascular disease than age-matched women. In the current studies, we found that cardiac hypertrophy and fibrosis were significantly more pronounced in males compared with females of guanylyl cyclase-A knockout (GC-A KO) mice at 16 wk of age. These gender-related differences were not seen in wild-type mice. In the further studies, either castration (at 10 wk of age) or flutamide, an androgen receptor antagonist, markedly attenuated cardiac hypertrophy and fibrosis in male GC-A KO mice without blood pressure change. In contrast, ovariectomy (at 10 wk of age) had little effect. Also, chronic testosterone infusion increased cardiac mass and fibrosis in ovariectomized GC-A mice. None of the treatments affected cardiac mass or the extent of fibrosis in wild-type mice. Overexpression of mRNAs encoding atrial natriuretic peptide, brain natriuretic peptide, collagens I and III, TGF-beta1, TGF-beta3, angiotensinogen, and angiotensin converting enzyme in the ventricles of male GC-A KO mice was substantially decreased by castration. The gender differences were virtually abolished by targeted deletion of the angiotensin II type 1A receptor gene (AT1A). Neither castration nor testosterone administration induced any change in the cardiac phenotypes of double-KO mice for GC-A and AT1A. Thus, we suggest that androgens contribute to gender-related differences in cardiac hypertrophy and fibrosis by a mechanism involving AT1A receptors and GC-A.

Androgen Receptor Antagonists↗

PubMed2003

A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets.

Understanding mammalian spermatozoan development and the events surrounding fertilization has grown slowly, in part because of uncertainty about the number and identity of the cellular components involved. Determination of those transcripts expressed specifically by germ cells should provide an inclusive list of probable critical proteins. Here, total mouse testis transcript profiles were trimmed of transcripts found in cultures enriched in Sertoli or interstitial cells to yield a germ cell-enriched transcript profile. Monitoring of changes of this profile in the developing testis identified 1,652 genes whose transcript abundance increased markedly coincident with the onset of meiosis. Remarkably, 351 of these genes (approximately equal to 20%) appear to be expressed only in the male germline. Germ cell-specific transcripts are much less common earlier in testis development. Further analysis of the UniGene EST database coupled with quantitative PCR indicates that approximately 4% of the mouse genome is dedicated to expression in postmeiotic male germ cells. Most or many of the protein products of these transcripts are probably retained in mature spermatozoa. Targeted disruption of 19 of these genes has indicated that a majority have roles critical for normal fertility. Thus, we find an astonishing number of genes expressed specifically by male germ cells late in development. This extensive group provides a plethora of potential targets for germ cell-directed contraception and a staggering number of candidate proteins that could be critical for fertilization.

Animals↗

PubMed2003

Regulation of the guanylyl cyclase-B receptor by alternative splicing.

Guanylyl cyclase-B (GC-B) is a single transmembrane receptor that binds C-type natriuretic peptide (CNP). The ligand/receptor appears critical in the regulation of cell proliferation and differentiation where it acts as an adversary of mitogenic signaling pathways. We have isolated three guanylyl cyclase-B isoforms generated from a single gene by alternative splicing and termed them GC-B1, GC-B2, and GC-B3. GC-B1 is full-length and responds maximally to CNP, GC-B2 contains a 25-amino acid deletion in the protein kinase homology domain, and GC-B3 only retains a part of the extracellular ligand-binding domain. GC-B2 binds CNP, but the ligand fails to activate the cyclase, while GC-B3 fails to bind ligand. When GC-B2 or GC-B3 is expressed coincident with GC-B1, they act as dominant negative isoforms by virtue of blocking formation of active GC-B1 homodimers. Relative expression levels of GC-B1, GC-B2, and GC-B3 vary across tissues and as a function of in vitro culture; the relative amount of GC-B2 to GC-B1 is repressed in cultured smooth muscle cells relative to endogenous ratios in the medial layer cells of the aorta. Thus, GC-B isoform levels can be independently regulated. Given that the splice variants serve as dominant negative forms, these will serve as regulators of the full-length GC-B.

Alternative Splicing↗

PubMed2003

Significance and therapeutic potential of the natriuretic peptides/cGMP/cGMP-dependent protein kinase pathway in vascular regeneration.

Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), are characterized as cardiac or vascular hormones that elicit their biological effects by activation of the cGMPcGMP-dependent protein kinase (cGK) pathway. We recently reported that adenoviral gene transfer of CNP into rabbit blood vessels not only suppressed neointimal formation but also accelerated reendothelialization, a required step for endothelium-dependent vasorelaxation and antithrombogenicity. Accordingly, we investigated the therapeutic potential of the NPscGMPcGK pathway for vascular regeneration. In transgenic (Tg) mice that overexpress BNP in response to hindlimb ischemia, neovascularization with appropriate mural cell coating was accelerated without edema or bleeding, and impaired angiogenesis by the suppression of nitric oxide production was effectively rescued. Furthermore, in BNP-Tg mice, inflammatory cell infiltration in ischemic tissue and vascular superoxide production were suppressed compared with control mice. Ischemia-induced angiogenesis was also significantly potentiated in cGK type I Tg mice, but attenuated in cGK type I knockout mice. NPs significantly stimulated capillary network formation of cultured endothelial cells by cGK stimulation and subsequent Erk12 activation. Furthermore, gene transfer of CNP into ischemic muscles effectively accelerated angiogenesis. These findings reveal an action of the NPscGMPcGK pathway to exert multiple vasculoprotective and regenerative actions in the absence of apparent adverse effects, and therefore suggest that NPs as the endogenous cardiovascular hormone can be used as a strategy of therapeutic angiogenesis in patients with tissue ischemia.

Animals↗

PubMed2003

Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A.

Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Atrial natriuretic peptide (ANP) has been postulated to exert local antihypertrophic effects in the heart. Thus, a loss of function of the ANP receptor guanylyl cyclase-A (GC-A) might contribute to the increased propensity to cardiac hypertrophy, although a causative role in vivo has not been definitively demonstrated. To test whether local ANP modulates cardiomyocyte growth, we inactivated the GC-A gene selectively in cardiomyocytes by homologous loxP/Cre-mediated recombination. Thereby we have circumvented the systemic, hypertensive phenotype associated with germline inactivation of GC-A. Mice with cardiomyocyte-restricted GC-A deletion exhibited mild cardiac hypertrophy, markedly increased mRNA expression of cardiac hypertrophy markers such as ANP (fivefold), alpha-skeletal actin (1.7-fold), and beta-myosin heavy chain (twofold), and increased systemic circulating ANP levels. Their blood pressure was 7-10 mmHg below normal, probably because of the elevated systemic levels and endocrine actions of ANP. Furthermore, cardiac hypertrophic responses to aortic constriction were enhanced and accompanied by marked deterioration of cardiac function. This phenotype is consistent with a local function of the ANP/GC-A system to moderate the molecular program of cardiac hypertrophy.

Actins↗

PubMed2002

Production of transgenic rats by lentiviral transduction of male germ-line stem cells.

Primary cultures of rat spermatogenic cells that did not bind to collagen matrices were able to colonize and form mature spermatozoa when transferred to testes of recipient males. Up to 73% of the progeny from matings with recipient males were derived from the transferred spermatogenic cells. Subsequently, two populations of germ cells were obtained by selection on laminin matrices. Both populations expressed the spermatogenic cell marker, DAZL, but not the somatic cell marker, vimentin. The cells that bound to laminin represented approximately 5% of the total population and were greatly enriched in ability to colonize a recipient testis, suggesting an enrichment in germ-line stem cells. The colonization potential was maintained for at least 7 days in culture. These cells were subsequently transduced with a lentiviral enhanced GFP reporter vector and then transferred to WT recipient males. After mating, 26 of 44 pups were derived from the cultured donor germ cells, and 13 pups carried the lentiviral transgene. Based on Southern analysis, the transgene was integrated at a different genetic locus in each animal and was transmitted to approximately 50% of pups in the F(2) generation. Thus, by using these procedures, approximately 30% of pups in the F(1) generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.

Amino Acid Sequence↗

PubMed2002

Guanylyl cyclase-A inhibits angiotensin II type 1A receptor-mediated cardiac remodeling, an endogenous protective mechanism in the heart.

BACKGROUND: Guanylyl cyclase (GC)-A, a natriuretic peptide receptor, lowers blood pressure and inhibits the growth of cardiac myocytes and fibroblasts. Angiotensin II (Ang II) type 1A (AT1A), an Ang II receptor, regulates cardiovascular homeostasis oppositely. Disruption of GC-A induces cardiac hypertrophy and fibrosis, suggesting that GC-A protects the heart from abnormal remodeling. We investigated whether GC-A interacts with AT1A signaling in the heart by target deletion and pharmacological blockade or stimulation of AT1A in mice. METHODS AND RESULTS: We generated double-knockout (KO) mice for GC-A and AT1A by crossing GC-A-KO mice and AT1A-KO mice and blocked AT1 with a selective antagonist, CS-866. The cardiac hypertrophy and fibrosis of GC-A-KO mice were greatly improved by deletion or pharmacological blockade of AT1A. Overexpression of mRNAs encoding atrial natriuretic peptide, brain natriuretic peptide, collagens I and III, transforming growth factors beta1 and beta3, were also strongly inhibited. Furthermore, stimulation of AT1A by exogenous Ang II at a subpressor dose significantly exacerbated cardiac hypertrophy and dramatically augmented interstitial fibrosis in GC-A-KO mice but not in wild-type animals. CONCLUSIONS: These results suggest that cardiac hypertrophy and fibrosis of GC-A-deficient mice are partially ascribed to an augmented cardiac AT1A signaling and that GC-A inhibits AT1A signaling-mediated excessive remodeling.

Angiotensin II↗

PubMed2002

Male germ cell specification and differentiation.

Understanding the mechanisms by which the germline is induced and maintained should lead to a broader understanding of the means by which pluripotency is acquired and maintained. In this review, two major aspects of male germ cell development are discussed: underlying mechanisms for induction and maintenance of primordial germ cells and the basic signaling pathways that determine spermatogonial cell fate.

Animals↗