16-Unsaturated C19 3-oxo steroids as metabolic intermediates in boar testis

1. The formation of the two 16-unsaturated alcohols 5α-androst-16-en-3α-ol and 5α-androst-16-en-3β-ol from [5α-3H]5α-androst-16-en-3-one has been demonstrated in boar testis homogenates. 2. The optimum yield (23%) of the 3α-alcohol was obtained in the presence of NADPH, whereas that for the 3β-alcohol (74%) was obtained when NADH was the added cofactor. 3. The two alcohols were not interconvertible. 4. Prolonged storage of boar testis tissue at −20°C abolished the ability to form all androst-16-enes except androsta-4,16-dien-3-one from [4-14C]progesterone. 5. The production of 5α-androst-16-en-3-one and the two alcohols from [7α-3H]androsta-4,16-dien-3-one only occurred when fresh tissue was used, whereas reduction of [5α-3H]5α-androst-16-en-3-one was unaffected by storage of testis at −20°C. 6. NADPH was the preferred cofactor for the reduction of androsta-4,16-dien-3-one. 7. The previously established conversion of androsta-5,16-dien-3β-ol into androsta-4,16-dien-3-one was shown to be reversible, NADH and NADPH being equally effective cofactors. 8. Pathways of biosynthesis of 5α-androst-16-en-3α- and 3β-ols, with the C19 3-oxo steroids as intermediates, are presented.

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Postchemotherapy Ejaculatory Azoospermia: Fatherhood With Sperm From Testis Tissue With Intracytoplasmic Sperm Injection

Journal of Clinical Oncology ◽

10.1200/jco.2002.20.4.930 ◽

2002 ◽

Vol 20 (4) ◽

pp. 930-936 ◽

Cited By ~ 27

Author(s):

M. N. Damani ◽

V. Masters ◽

M. V. Meng ◽

C. Burgess ◽

P. Turek ◽

...

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Fertilization Rate ◽

Nonobstructive Azoospermia ◽

Fresh Tissue ◽

Icsi Cycle ◽

Small Subgroup ◽

History Of ◽

Fertility Potential ◽

Late Maturation ◽

Testis Tissue

PURPOSE: To define the success of testis sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) in azoospermic men with a history of chemotherapy. PATIENTS AND METHODS: In a retrospective study, 23 men with ejaculatory azoospermia and a history of chemotherapy underwent TESE in a search for usable spermatozoa. In six patients cryopreserved tissue and in nine patients fresh tissue provided sperm for an ICSI cycle. Histologic analysis of the testis was performed in all patients. The presence or absence of sperm, fertilization rates with ICSI, and final outcomes of pregnancy were recorded. RESULTS: Spermatozoa were found on TESE in 15 (65.2%) of 23 men. On histopathology, the predominant pattern observed was Sertoli cell only (47.8%), followed by hypospermatogenesis (30.4%), mixed (17.4%), and late maturation arrest (4.3%). The fertilization rate was 65.2%, and ongoing/delivered pregnancies occurred in 30.8% of cycles. Six healthy boys and four healthy girls have been born to date. CONCLUSION: Men who are azoospermic and have had prior cytotoxic therapy make up a small subgroup of males with nonobstructive azoospermia. It is important to define and characterize this subgroup and better define their true fertility potential. Approximately two thirds of these men have retrievable testis sperm, which may be used with ICSI to have healthy offspring. This exciting avenue for paternity has heretofore not been available to such patients.

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The biosynthesis of androst-16-enes in boar testis tissue

Biochemical Journal ◽

10.1042/bj1170533 ◽

1970 ◽

Vol 117 (3) ◽

pp. 533-538 ◽

Cited By ~ 31

Author(s):

Tatyana Katkov ◽

D. B. Gower

Keyword(s):

Short Term ◽

Fresh Tissue ◽

Labelled Compound ◽

Testis Tissue

1. The metabolism of [4-14C]pregnenolone to androst-16-enes has been studied in short-term incubations of boar testis tissue. With fresh tissue androsta-5,16-dien-3β-ol (8%) and 5α-androst-16-en-3β-ol (2%) were formed. Tissue that had been stored at −20°C was still capable of metabolizing pregnenolone to androsta-5,16-dien-3β-ol. 2. NADPH was essential for the formation of androsta-5,16-dien-3β-ol from pregnenolone; NADH had less activity and ATP was not necessary for the reaction. 3. [4-14C]Androsta-5,16-dien-3β-ol, prepared biosynthetically from [4-14C]pregnenolone, was shown to be converted by boar testis preparations into androsta-4,16-dien-3-one (31%) if NAD+ was present or into 5α-androst-16-en-3β-ol (4%) if NADPH was present. 4. 17α-Hydroxyandrost-4-en-3-one and 3β,17α-dihydroxypregn-5-en-20-one were considered as possible precursors for androst-16-ene formation, but both were shown to be ineffective. 5. No radioactivity was incorporated into androst-5-en-3β-ol used to trap any corresponding 14C-labelled compound formed from [4-14C]pregnenolone.

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Spermatogenesis and steroidogenesis in mouse, hamster and monkey testicular tissue after cryopreservation and heterotopic grafting to castrated hosts

Reproduction ◽

10.1530/rep.0.1240339 ◽

2002 ◽

pp. 339-346 ◽

Cited By ~ 157

Author(s):

S Schlatt ◽

SS Kim ◽

R Gosden

Keyword(s):

Adult Mouse ◽

Germ Line ◽

Testicular Tissue ◽

Seminiferous Tubules ◽

Male Germ Line ◽

Fresh Tissue ◽

Intact Control ◽

Adult Mice ◽

Mature Spermatozoa ◽

Testis Tissue

Retrieval, extracorporal storage and autotransplantation of testicular tissue could become an important strategy for preserving male gonadal function. The present study used syngeneic and immunodeficient nude mice as hosts, and immature and adult mice, neonatal and adult photoregressed Djungarian hamsters and neonatal marmosets to identify the potential of testicular tissue grafting to maintain the morphological and functional integrity of the testis. Testicular tissue was grafted s.c. either as fresh tissue or after cryopreservation into adult, orchidectomized hosts. The mice that received rodent testis tissue were autopsied 50 days later, and blood samples were collected. Sixty-five per cent of mouse isografts contained morphologically normal testicular tissue and seminiferous tubules with some degree of spermatogenic recovery. Mature spermatozoa were recovered after enzymatic disaggregation. Although the recovery of spermatogenesis was limited in adult mouse and hamster tissue, complete spermatogenesis was observed in grafts from immature rodents. Testicular tissue from neonatal marmosets developed up to the stage of spermatocytes at day 135 after xenografting. Androgen concentrations were comparable in intact control mice and in mice receiving fresh mouse and hamster grafts, slightly lower in mice receiving cryopreserved grafts and adult photoregressed hamster tissue, and low in castrated control mice and in mice receiving marmoset tissue. These results show that isografts and xenografts of immature and adult testicular tissue become functionally active as a s.c. graft in the mouse and that this approach might be useful in combination with cryopreservation as a tool for storage and activation of the male germ line and androgen replacement therapy in patients.

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SEM Cold Stage Techniques for the Observation of Vegetative and Floral Apices of Oat (Avena sativa L.) Plants

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100080377 ◽

1977 ◽

Vol 35 ◽

pp. 590-591

Author(s):

B. K. Kirchoff ◽

L.F. Allard ◽

W.C. Bigelow

Keyword(s):

Critical Point ◽

Avena Sativa ◽

Substantial Improvement ◽

Fresh Tissue ◽

Cold Stage ◽

Critical Point Drying ◽

Almost All ◽

Cell Collapse ◽

Conductive Paint ◽

Carbon Based

In attempting to use the SEM to investigate the transition from the vegetative to the floral state in oat (Avena sativa L.) it was discovered that the procedures of fixation and critical point drying (CPD), and fresh tissue examination of the specimens gave unsatisfactory results. In most cases, by using these techniques, cells of the tissue were collapsed or otherwise visibly distorted. Figure 1 shows the results of fixation with 4.5% formaldehyde-gluteraldehyde followed by CPD. Almost all cellular detail has been obscured by the resulting shrinkage distortions. The larger cracks seen on the left of the picture may be due to dissection damage, rather than CPD. The results of observation of fresh tissue are seen in Fig. 2. Although there is a substantial improvement over CPD, some cell collapse still occurs.Due to these difficulties, it was decided to experiment with cold stage techniques. The specimens to be observed were dissected out and attached to the sample stub using a carbon based conductive paint in acetone.

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Prolonged Fixation Studies For Spaceflight

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100072101 ◽

1973 ◽

Vol 31 ◽

pp. 332-333

Author(s):

Robert Corbett ◽

Delbert E. Philpott ◽

Sam Black

Keyword(s):

High Pressure ◽

Living Systems ◽

Prolonged Storage ◽

Time Intervals ◽

Early Signs ◽

Large Numbers

Observation of subtle or early signs of change in spaceflight induced alterations on living systems require precise methods of sampling. In-flight analysis would be preferable but constraints of time, equipment, personnel and cost dictate the necessity for prolonged storage before retrieval. Because of this, various tissues have been stored in fixatives and combinations of fixatives and observed at various time intervals. High pressure and the effect of buffer alone have also been tried.Of the various tissues embedded, muscle, cartilage and liver, liver has been the most extensively studied because it contains large numbers of organelles common to all tissues (Fig. 1).

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Application of EM to differentiate herpes virus from pox virus in genital specimens

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169043 ◽

1994 ◽

Vol 52 ◽

pp. 262-263

Author(s):

K. Rekrut ◽

K. Schleuter

Keyword(s):

Tissue Culture ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Fresh Tissue ◽

Culture Cells ◽

Viral Transport ◽

Carbon Coated ◽

Immuno Assay ◽

Simplex Virus ◽

Enzyme Immuno Assay

Confirmation of herpes simplex virus (HSV) from genital lesions of obstetrical (OB) patients may affect both the management of the delivery and of the neonate.(l,2) During 1992 and 1993, 4,450 genital specimens from OB patients were submitted in viral transport media for herpes culture. The specimens were inoculated into MRC-5, Vero, and A-549 tissue culture tubes, incubated, and examined daily for 7 days for cytopathic effect (CPE). The original specimens were frozen at −70° C until final reports were issued. Culture tubes with CPE were tested by the Dupont Herpchek enzyme immuno assay (EIA) to confirm the presence of herpes simplex virus (HSV). (3,4) 170 OB patient specimens were positive by culture and confirmed by EIA.There were also 63 cultures exhibiting CPE ressembling HSV which were negative by EIA testing, which failed to pass in fresh tissue culture cells or progress to more enhanced CPE in culture. These original specimens were screened by electron microscopy after direct ultracentrifugation employing the Beckman airfuge with the EM 90 rotor on to formvar carbon-coated 300 mesh copper grids and negatively stained with 2% PTA.

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Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity

Biochemical Journal ◽

10.1042/bcj20190427 ◽

2019 ◽

Vol 476 (16) ◽

pp. 2297-2319 ◽

Cited By ~ 3

Author(s):

Marta Grzechowiak ◽

Milosz Ruszkowski ◽

Joanna Sliwiak ◽

Kamil Szpotkowski ◽

Michal Sikorski ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Size Exclusion ◽

Inorganic Pyrophosphatase ◽

Prolonged Storage ◽

Mitochondrial Targeting ◽

Cleavage Rate ◽

Inorganic Pyrophosphate ◽

Putative Signal Peptide ◽

Targeting Peptide

Abstract Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31–Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

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HIF1-α-Mediated Gene Expression Induced by Vitamin B1 Deficiency

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000159 ◽

2013 ◽

Vol 83 (3) ◽

pp. 188-197 ◽

Cited By ~ 21

Author(s):

Rebecca L. Sweet ◽

Jason A. Zastre

Keyword(s):

Gene Expression ◽

Thiamine Deficiency ◽

Glucose Transporter ◽

Neuroblastoma Cell ◽

Hypoxia Inducible Factor ◽

Clinical Manifestations ◽

Vitamin B1 ◽

Low Oxygen ◽

Glucose Transporter 1 ◽

Metabolic Intermediates

It is well established that thiamine deficiency results in an excess of metabolic intermediates such as lactate and pyruvate, which is likely due to insufficient levels of cofactor for the function of thiamine-dependent enzymes. When in excess, both pyruvate and lactate can increase the stabilization of the hypoxia-inducible factor 1-alpha (HIF-1α) transcription factor, resulting in the trans-activation of HIF-1α regulated genes independent of low oxygen, termed pseudo-hypoxia. Therefore, the resulting dysfunction in cellular metabolism and accumulation of pyruvate and lactate during thiamine deficiency may facilitate a pseudo-hypoxic state. In order to investigate the possibility of a transcriptional relationship between hypoxia and thiamine deficiency, we measured alterations in metabolic intermediates, HIF-1α stabilization, and gene expression. We found an increase in intracellular pyruvate and extracellular lactate levels after thiamine deficiency exposure to the neuroblastoma cell line SK-N-BE. Similar to cells exposed to hypoxia, there was a corresponding increase in HIF-1α stabilization and activation of target gene expression during thiamine deficiency, including glucose transporter-1 (GLUT1), vascular endothelial growth factor (VEGF), and aldolase A. Both hypoxia and thiamine deficiency exposure resulted in an increase in the expression of the thiamine transporter SLC19A3. These results indicate thiamine deficiency induces HIF-1α-mediated gene expression similar to that observed in hypoxic stress, and may provide evidence for a central transcriptional response associated with the clinical manifestations of thiamine deficiency.

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