PubMed Health⌕ Search

Biomedical subjects

Kim Boekelheide

Publications and source records attributed to Kim Boekelheide.

At least 19 recordsLinked to original sources
PubMed2006

NF-kappaB activation elicited by ionizing radiation is proapoptotic in testis.

The transcription factor NF-kappaB modulates apoptotic machinery following activation by the IkappaB kinase (IKK) complex. Inhibiting activity of one of the catalytic subunits of the IKK complex, IKKbeta (also known as IKBKB and IKK2) severely inhibits NF-kappaB nuclear translocation in response to most stimuli, including ionizing radiation. Doubly floxed Ikkbeta(F/F) mice (control) were compared to haplo-insufficient Ikkbeta(F/)(delta) mice (NF-kappaB knockdown) to examine the in vivo apoptotic role of NF-kappaB in the testis. Although Ikkbeta(F/F) control adult mice had spermatid head counts and testis and body weights similar to Ikkbeta(F/)(delta) mice, cellular stress in the form of ionizing radiation elicited a differential phenotype. Lower body exposure to 5 Gy of ionizing radiation induced a greater NF-kappaB activation in Ikkbeta(F/F) than in Ikkbeta(F/)(delta) mice. In addition, exposure to ionizing radiation resulted in fewer apoptotic germ cells 3, 6, and 12 h after injury in NF-kappaB knockdown mice than in controls, concomitant with the reduced cleavage of caspases 3 and 9 at 3 h. There was also a reduction in total germ cells lost after radiation with NF-kappaB inhibition. Correspondingly, real-time RT-PCR showed a significant reduction in Cdnk1a (also known as p21) and Fasl expression 3 and 6 h, respectively, after irradiation in Ikkbeta(F/)(delta) compared to control testes. These data indicate that, despite acting in an antiapoptotic manner in many tissue types, NF-kappaB is proapoptotic in modulating the germ cell response to ionizing radiation.

Animals↗
PubMed2006

Extensive neuroprotection by choroid plexus transplants in excitotoxin lesioned monkeys.

Huntington's disease (HD) results from degeneration of striatal neurons. Choroid plexus (CP) cells secrete neurotrophic factors, and CP transplants are neuroprotective in rat models of HD. To determine if similar neuroprotective effects could be obtained in primates, porcine CP was encapsulated in alginate capsules. PCR confirmed that the CP cells expressed transthyretin and immunocytochemistry demonstrated typical ZO-1 and tubulin staining. In vitro, CP conditioned media enhanced the survival and preserved neurite number and length on serum deprived neurons. Cynomolgus primates were transplanted with CP-loaded capsules into the caudate and putamen followed by quinolinic acid (QA) lesions 1 week later. Control monkeys received empty capsules plus QA. Choroid plexus transplants significantly protected striatal neurons as revealed by stereological counts of NeuN-positive neurons (8% loss vs. 43% in controls) and striatum volume (10% decrease vs. 40% in controls). These data indicate that CP transplants might be useful for preventing the degeneration of neurons in HD.

Animals↗
PubMed2006

The Sycp1-Cre transgenic mouse and male germ cell inhibition of NF-kappa b.

Successful spermatogenesis requires autocrine, paracrine, and endocrine signaling throughout the testes. The seminiferous tubules contain somatic Sertoli cells in tight association with numerous germ cell populations. To address the in vivo biologic roles of genes during spermatogenesis, spatial and temporal restriction of gene inhibition is a useful approach. To this end, Cre-LoxP technology can produce cell-specific knockdowns of genes, allowing dissection of the underlying processes that manifest as functional deficits in whole animals. Here we report the use of the synaptonemal complex protein 1-Cre (Sycp1-Cre) to create germ cell-specific nuclear factor kappaB knockdown mice through floxed IkappaB kinasebeta. We observed a LoxP gene recombination rate of approximately 43% using Sycp1-Cre, as determined by offspring genotype. In addition, we confirm that, with multiple generations, the LoxP sites fail to recombine due to epigenetic modification. This detailed examination of the meiotic Sycp1-Cre recombinase activity highlights the obstacles to germ cell-specific gene inhibition through Cre/LoxP technology in the testis. Taken together, these data demonstrate a need for early spermatogonial expression of Cre recombinase, as an alternative to meiotic Cre expression, for the creation of germ cell-specific knockout mice.

Animals↗
PubMed2005

Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat Sertoli cells and disrupts Sertoli cell-gonocyte contact.

Di(n-butyl) phthalate (DBP) is commonly used in personal care products and as a plasticizer to soften consumer plastic products. Male rats exposed to DBP in utero have malformations of the male reproductive tract and testicular atrophy characterized by degeneration of seminiferous epithelium and decreased sperm production. In the fetal testis, in utero exposure to DBP reportedly resulted in reduced testosterone levels, Leydig cell aggregates, and multinucleated gonocytes (MNG). We investigated whether exposure in utero to DBP affects rat fetal Sertoli cells and compromises interactions between Sertoli and germ cells in the developing testis. Histological examination showed that MNG occurred at low frequency in the normal fetal rat testis. Exposure in utero at the dose level of DBP above estimated environmental or occupational human exposure levels significantly increased the number of these abnormal germ cells. Postnatally, MNG exhibited aberrant mitoses and were detected at the basal lamina. MNG were not apoptotic in the fetal and postnatal rat testes, as indicated by TUNEL. Sertoli cells in DBP-exposed fetal testis had retracted apical processes, altered organization of the vimentin cytoskeleton, and abnormal cell-cell contacts with gonocytes. The effect of DBP on Sertoli cell morphology at the level of light microscopy was reversed after birth and cessation of exposure. Our data indicate that fetal Sertoli cells are targeted by exposure in utero to DBP and suggest that abnormal interactions between Sertoli and germ cells during fetal life play a role in the development of MNG.

Animals↗
PubMed2005

Mechanisms of toxic damage to spermatogenesis.

Azoospermia and long-lasting testicular atrophy are common adverse consequences of cancer treatment. Chemotherapeutic agents may disrupt spermatogenesis by targeting various testicular cell types (Leydig cells, Sertoli cells, and germ cells) and by activating numerous molecular pathways involved in germ cell life-and-death decision making. Genetically modified animal models with deficiencies in specific proapoptotic and prosurvival pathways have become powerful tools in understanding the molecular regulation of spermatogenesis and the response of the seminiferous epithelium to toxic injury. In this brief review, selected examples of results of toxic exposures in genetically deficient animal models are discussed to highlight the roles of p53 and the Fas system as modulators of proapoptotic activity in the testis. A final section focuses on cisplatin, a cancer chemotherapeutic agent that produces male reproductive toxicity by targeting multiple cell types in the testis.

Antineoplastic Agents↗
PubMed2005

Testicular microlithiasis in association with pseudoxanthoma elasticum.

PURPOSE: To determine the presence of testicular microlithiasis in male subjects with pseudoxanthoma elasticum (PXE). MATERIALS AND METHODS: Institutional review board approval was obtained for the prospective and retrospective components of this HIPAA-compliant study. Informed consent was obtained from all patients or their parents. Testicular ultrasonography (US) was performed in eight men aged 29-56 years and in one 13-year-old boy, all with confirmed PXE. Two radiologists reviewed the US images by consensus for testicular microlithiasis, testicular masses, and additional testicular abnormalities. Testicular microlithiasis was judged to be classic when at least five microliths were seen on a single US image and to be limited when fewer than five microliths were seen on all obtained US images. Urologic physiologic examinations were performed. A history and/or symptoms of testicular disease also were recorded at the time of examination. Similarly, the testicular US images obtained in two additional men, aged 48 and 59 years, and in another 13-year-old boy were retrospectively reviewed. Histopathologic testicular analysis was performed in one autopsy case. RESULTS: Of the 12 participants, 11 (92%) had classic and one (8%) had limited testicular microlithiasis. None of the 12 participants had evidence of testicular malignancy at US or physical examination. Histopathologic analysis at autopsy revealed intratubular microlithiasis without the calcification of elastic fibers in arterial walls that is characteristic of cutaneous PXE. CONCLUSION: Study findings suggested an association between PXE and testicular microlithiasis. It is possible that the testicular microlithiasis in male subjects who have PXE is related to the underlying PXE abnormality.

Adolescent↗
PubMed2004

NF-kappaB is activated in the rat testis following exposure to mono-(2-ethylhexyl) phthalate.

The process of spermatogenesis requires a delicate balance of proapoptotic and antiapoptotic signaling to maintain optimal sperm output. A major transcription factor known to regulate numerous apoptosis-related genes is NF-kappaB. Here we show that mono-(2-ethylhexyl) phthalate (MEHP, 1 g/kg) induces translocation of NF-kappaB subunits (p65, p50, and c-Rel) to germ cell nuclei in young rats (Postnatal Day 28) as early as 1 h after exposure. Immunohistochemistry of rat testes exposed to MEHP showed increased p50 and c-Rel presence in spermatocytes and spermatogonia. In addition, there was increased p65 nuclear positivity in Sertoli cells and germ cells after MEHP, while Rel-B localization was unchanged. These alterations correlated with increased nuclear NF-kappaB-binding activity after MEHP exposure, as shown by electrophoretic mobility shift assays of whole-testis nuclear protein extracts. The increased activity of this transcription factor was associated with a transient protection of the seminiferous epithelium manifested as a decreased number of germ cell apoptotic nuclei measured by TUNEL assay 6 h after MEHP exposure. These results suggest that NF-kappaB is involved in the testicular response to MEHP-induced injury.

Animals↗
PubMed2004

Xenobiotic and endobiotic transporter mRNA expression in the blood-testis barrier.

A major function of xenobiotic and endobiotic transporters is to move a wide range of organic substances across cell membranes. Sertoli cells play an important role in protecting developing germ cells by forming a physiological barrier, limiting exposure to potentially toxic substrates, or conversely, facilitating uptake of xenobiotics within the testis. The aim of this study was to quantitatively determine the constitutive expression of various transporters in isolated Sertoli cells from adult Sprague-Dawley rats. The following mRNA levels were measured in isolated Sertoli cells by the branched DNA signal amplification method, multidrug resistance (Mdr) protein 1a, 1b, and 2; multiple drug resistance protein (Mrp) 1, 2, 3, 4, 5, 6, 7, and 8; sodium taurocholate cotransporting polypeptide; bile salt excretory protein; ileal bile acid transporter; AbcG5 and AbcG8; organic anion transporting polypeptide (Oatp) 1, 2, 3, 4, 5, 9, and 12; prostaglandin transporter (Pgt); testis-specific transporter (Tst) 1 and Tst2; organic anion transporter (Oat) 1, 2, 3, and K; organic cation transporter (Oct) 1, 2, 3, N1, and N2; divalent metal transporter (Dmt) 1, Menke's, and Wilson's; zinc transporter (Znt) 1; equilibrative nucleoside transporter (Ent) 1 and 2; concentrative nucleoside transporter (Cnt) 1 and 2; and peptide transporter (Pept) 1 and 2. Levels were also determined in whole testis, liver, kidney, and ileum to provide a reference for determining relative expression levels. Mrp8, Tst1 and 2, and Ent1 and 2 were expressed in Sertoli cells at higher levels than in liver, kidney, or ileum, whereas Mrp1, 5, and 7, Mdr2, Oatp3, Oat2, OctN2, Dmt1, Menke's, Wilson's, and Znt1 were all significantly expressed in Sertoli cells, but Sertoli cell expression was not the tissue of highest expression. The remaining transporters were expressed at low levels in isolated Sertoli cells. Additionally, expression levels of Mrp1, Mrp7, Mrp8, Tst1, Tst2, OctN2, Wilson's, Znt1, Ent1, and Ent2 were greater in isolated Sertoli cells than in whole testis. Constitutive expression of transporters in Sertoli cells may provide an insight into the range of xenobiotics that can potentially be transported by Sertoli cells and thereby provide a mechanistic under standing of blood-testis barrier function.

Animals↗
PubMed2004

2,5-hexanedione and carbendazim coexposure synergistically disrupts rat spermatogenesis despite opposing molecular effects on microtubules.

2,5-Hexanedione (2,5-HD), a taxol-like promoter of microtubule assembly, and carbendazim (CBZ), a colchicine-like inhibitor of microtubule assembly, are two environmental testicular toxicants that target and disrupt microtubule function in Sertoli cells. At the molecular level, these two toxicants have opposite effects on microtubule assembly, yet they share the common physiologic effect of inhibiting microtubule-dependent functions of Sertoli cells. By studying a combined exposure to 2,5-HD and CBZ, we sought to determine whether CBZ would antagonize or exacerbate the effects of an initial 2,5-HD exposure. In vitro, 2,5-HD-treated tubulin had a decreased lag time and an increased maximal velocity of microtubule assembly. These 2,5-HD-induced in vitro alterations in microtubule assembly were normalized by CBZ exposure. In vivo, adult male rats were exposed to a 1% solution of 2,5-HD in the drinking water for 2.5 weeks. CBZ was administered by gavage (200 mg/kg body weight) at the same time as unilateral surgical ligation of the efferent ducts, 24 h before evaluation of the testis. Measures of testicular effect (testis weight, histopathologic changes [sloughing and vacuolization], and seminiferous tubule diameters) were all significantly altered with combined exposure. The testicular effects in the combined exposure group were either different (seminiferous tubule diameters), additive (% vacuolization), or greater than additive (% sloughing) compared to the effects of the individual toxicant exposure groups referenced to the controls. Therefore, CBZ coexposure does not antagonize the effects of an initial 2,5-HD exposure, as might be expected if their molecular effects on microtubule assembly were solely responsible for their combined toxicity; instead, 2,5-HD and CBZ act together to exacerbate the testicular injury.

Animals↗
PubMed2003

gamma-Tubulin overexpression in Sertoli cells in vivo: I. Localization to sites of spermatid head attachment and alterations in Sertoli cell microtubule distribution.

Sertoli cells play a number of roles in supporting spermatogenesis, including structural organization, physical and paracrine support of germ cells, and secretion of factors necessary for germ cell development. Studies with microtubule disrupting compounds indicate that intact microtubule networks are crucial for normal spermatogenesis. However, treatment with toxicants and pharmacologic agents that target microtubules lack cell-type selectivity and may therefore elicit direct effects on germ cells, which also require microtubule-mediated activities for division and morphological transformation. To evaluate the importance of Sertoli cell microtubule-based activities for spermatogenesis, an adenoviral vector that overexpresses the microtubule nucleating protein, gamma-tubulin, was used to selectively disrupt microtubule networks in Sertoli cells in vivo. gamma-Tubulin overexpression was observed to cause redistribution of Sertoli cell microtubule networks, and overexpression of a gamma-tubulin-enhanced green fluorescent protein fusion protein was observed to localize to the site of elongate spermatid head attachment to the seminiferous epithelium.

Animals↗
PubMed2003

gamma-Tubulin overexpression in Sertoli cells in vivo. II: Retention of spermatids, residual bodies, and germ cell apoptosis.

The degree of germ cell dependence on Sertoli cell-mediated activities has been a subject of considerable attention. Sertoli cell secretory pathways have been extensively studied both in an effort to understand their normal physiologic roles and as targets for pharmacologic and toxicant activity. To determine the degree to which normal spermatogenesis depends on key functions of the Sertoli cell microtubule network, adenoviral vectors that overexpress the microtubule nucleating protein, gamma-tubulin, were delivered to Sertoli cells in vivo. gamma-Tubulin overexpression disrupts the Sertoli cell microtubule network (as described in the companion article); leads to gross disorganization of the seminiferous epithelium, inducing retention of spermatids and residual bodies; and causes germ cell apoptosis. These data are consistent with earlier studies in which toxicants and pharmacologic agents were used to disrupt microtubule networks. These data confirm that Sertoli cell microtubule networks play an important role in maintaining the organization of the seminiferous epithelium and that in the absence of an intact Sertoli cell microtubule network, germ cell viability is impaired.

Adenoviridae↗
PubMed2003

Expression of a K48R mutant ubiquitin protects mouse testis from cryptorchid injury and aging.

Testis injury models can be useful for determining the in vivo function of genes. In this study, ubiquitin, a tag for 26S-proteasome degradation, was mutated at lysine 48 (K48R) to inhibit ubiquitin chain assembly. K48R transgenic mice had testes with delayed germ cell loss following the acute injury of experimental cryptorchidism, and were resistant to the chronic injury of aging-associated testicular atrophy. After 4 days of cryptorchid-mediated heat stress, the average weight of cryptorchid testes in wild-type ubiquitin mice was significantly lower (P < 0.05) than in K48R mutant ubiquitin mice, indicating that altered ubiquitination delayed germ cell death. Light microscopy confirmed that the testicular injury, in both wild-type and K48R ubiquitin mice, was due to germ cell death. In addition, wild-type ubiquitin mice aged 19 to 22 months showed greater testicular atrophy and decreased average seminiferous tubule diameter when compared with K48R-aged testes. These results demonstrate a resistance to testicular injury conferred by the K48R mutation, suggesting that ubiquitin-mediated protein degradation is involved in the processing or modulation of testicular insults.

Aging↗
PubMed2003

The mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis.

The yeast Sir2p protein has an essential role in maintaining telomeric and mating type genes in their transcriptionally inactive state. Mammalian cells have a very large proportion of their genome inactive and also contain seven genes that have regions of homology with the yeast sir2 gene. One of these mammalian genes, sir2alpha, is the presumptive mammalian homologue of the yeast sir2 gene. We set out to determine if sir2alpha plays a role in mammalian gene silencing by creating a strain of mice carrying a null allele of sir2alpha. Animals carrying two null alleles of sir2alpha were smaller than normal at birth, and most died during the early postnatal period. In an outbred background, the sir2alpha null animals often survived to adulthood, but both sexes were sterile. We found no evidence for failure of gene silencing in sir2alpha null animals, suggesting that either SIR2alpha has a different role in mammals than it does in Saccharomyces cerevisiae or that its role in gene silencing in confined to a small subset of mammalian genes. The phenotype of the sir2alpha null animals suggests that the SIR2alpha protein is essential for normal embryogenesis and for normal reproduction in both sexes.

Animals↗
PubMed2002

2,5-hexanedione-induced testicular injury.

Now in its third decade of mechanistic investigation, testicular injury caused by 2,5-hexanedione (2,5-HD) exposure is a well-studied model with a rich database. The development of this model reflects the larger changes that have moved biology from a branch of chemistry into the molecular age. Critically examined in this review is the proposed mechanism for 2,5-HD-induced testicular injury in which germ cell maturation is disrupted owing to alterations in Sertoli cell microtubule-mediated functions. The goal is to evaluate the technical and conceptual approaches used to assess 2,5-HD-induced testicular injury, to highlight unanswered questions, and to identify fruitful avenues of future research.

Animals↗
PubMed2002

Dynamic testicular adhesion junctions are immunologically unique. I. Localization of p120 catenin in rat testis.

In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact, but the molecular composition of such junctions is not well known. At prototypical adherens junctions, proteins termed catenins bind to the intracellular domain of classic cadherins and regulate the strength of adhesion. Using a panel of monoclonal antibodies (5A7, 8D11, and 15D2), p120 catenin (p120) was localized in postnatal and adult rat testis cryosections and touch preparations by immunofluorescence. Immunoprecipitation of testis homogenates showed that at least four p120 isoforms were expressed from Postnatal Day 7 through adulthood. Both inter-Sertoli and Sertoli-germ cell junctions were p120-positive, however, individual p120 monoclonals were localized to specific junctions. The 5A7 and 8D11 antibodies colocalized with beta-catenin and plectin at inter-Sertoli and Sertoli-spermatocyte junctions. At inter-Sertoli junctions, p120 was juxtaposed to but did not colocalize with f-actin. Thus, p120 is likely a component of inter-Sertoli desmosome-like junctions. In contrast, the 15D2 monoclonal antibody specifically immunostained Sertoli-round spermatid and inter-Sertoli cell junctions in a dynamic pattern. From the time that round spermatids form to their differentiation into elongate spermatids, Sertoli-round spermatid 15D2 immunostaining cycled from a single mass to a curvilinear pattern, and finally to punctate structures scattered throughout the epithelium. This localization and stage-specific immunostaining pattern indicated that 15D2 recognized Sertoli-round spermatid desmosome-like junctions. Between Sertoli cells, 15D2 immunostained newly formed junctions (at Postnatal Days 21 through 43), but not mature junctions in the adult. From these data, we conclude that p120 is a component of most, if not all, desmosome-like junctions, and that desmosome-like junctions between different cell types contain a unique molecular composition.

Actins↗
PubMed2002

Dynamic testicular adhesion junctions are immunologically unique. II. Localization of classic cadherins in rat testis.

In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact and likely transmit signals between contacting cells. Defining the molecular composition of testicular cell-cell junctions is an important step in determining their function. Proteins belonging to the cadherin superfamily are important mediators of cell-cell adhesion, as well as cell signaling. Here, we determined the spatial and temporal protein expression of four classic cadherins in rat testis: N-cadherin, cadherin-6, cadherin-11, and a cadherin defined by an antiserum generated against a conserved classic cadherin peptide (L4). Through Western blot analysis, all antibodies recognized unique proteins. Similarly, each cadherin displayed unique, cell-type specific immunostaining patterns. Whereas N-cadherin, cadherin-11, and L4-positive cadherin were expressed from Postnatal Day 7 through adulthood, cadherin-6 protein was not present at Postnatal Day 7 and first appeared at Day 21. Immunostaining of testis cryosections on Postnatal Days 7, 21, 31, 43, and those of adults indicated that cadherin-11 localized to peritubular cell junctions. N-cadherin immunostaining localized to basal inter-Sertoli junctions, Sertoli-spermatocyte junctions, and at about stages I-VII in Sertoli-elongate spermatid junctions. Cadherin-6 immunostaining was restricted to Sertoli-round spermatid and in Sertoli-elongate spermatid junctions at approximately stages XII-I. Finally, L4-positive immunostaining also detected Sertoli-round spermatid junctions in addition to Sertoli-elongate spermatid junctions at approximately stages XII-I. These data show that the various testicular cell-cell junctions are molecularly unique and dynamic complexes.

Animals↗