Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.     

Structural requirements for the binding of prostaglandins

The binding of prostaglandins and analogs to the lipocyte PGE receptor was shown to exhibit a high degree of structural specificity. Small changes, particularly at the 9-keto or 15-hydroxy position, were found to drastically diminish interaction with the receptor. Studies of a rather substantial number of compounds revealed a close relationship between affinity for the lipocyte PGE receptor and the ability to stimulate cyclic AMP synthesis in the isolated mouse ovary. In general, activities in these two parameters follow the biological potencies generally recognized for these compounds.     

Apoptosis in C3H-10T1/2 cells: Roles of intracellular pH,protein kinase C,and the Na+/H+ antiporter

Changes in intracellular ion concentrations have been correlated with the activation of an endogenous endonuclease and thus internucleosomal DNA cleavage during apoptosis in many cell types. We investigated whether intracellular pH could play a significant role in apoptotic initiation and progression in C3H-10T1/2 cells, a cell strain that does not exhibit double-stranded DNA cleavage during apoptosis. Protein kinase C and the Na⁺/H⁺ antiporter, known regulators of intracellular pH, also were assessed for their involvement in apoptosis of C3H-10T1/2 cells. When a H⁺ ionophore was used to clamp intracellular pH to 6.0 or below, a significant level of apoptosis was induced in these cells within 6 h, whereas clamping at pH 6.75 did not induce significant amounts of apoptosis until 36 h after acidification. The acidified cells exhibited classic apoptotic morphology and chromatin condensation, similar to serum withdrawn cells, but failed to show internucleosomal DNA cleavage with electrophoresis of genomic DNA. Our results also suggest that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated inhibition of apoptosis in serum withdrawn C3H-10T1/2 cells functions through a sequential activation of protein kinase C and the Na⁺/H⁺ antiporter; thus, an alkalinization or an inhibition of acidification is involved in this apoptotic block. Serum withdrawal itself does not appear to act through a negative effect on either protein kinase C or the Na⁺/H⁺ antiporter. TPA was also capable of inhibiting the apoptosis induced by specific inhibitors of protein kinase C and the Na⁺/H⁺ antiporter, but the inhibition was successful only if the TPA was administered at least 20 min prior to the addition of the enzyme inhibitor. These results indicate that apoptosis in C3H-10T1/2 cells follows a pathway that involves intracellular acidification, but is independent of detectable endonuclease activity. J. Cell. Biochem. 67:231–240, 1997. © 1997 Wiley-Liss, Inc.     

Adaptation of subtype a human immunodeficiency virus type 1 envelope to pig-tailed macaque cells

The relevance of simian/human immunodeficiency virus (SHIV) infection of macaques to HIV-1 infection in humans depends on how closely SHIVs mimic HIV-1 transmission, pathogenesis, and diversity. Circulating HIV-1 strains are predominantly subtypes C and A and overwhelmingly require CCR5 for entry, yet most SHIVs incorporate CXCR4-using subtype B envelopes (Envs). While pathogenic subtype C-based SHIVs have been constructed, the subtype A-based SHIVs (SHIV-As) constructed to date have been unable to replicate in macaque cells. To understand the barriers to SHIV-A replication in macaque cells, HIVA(Q23)/SIV(vif) was constructed by engineering a CCR5-tropic subtype A provirus to express SIV vif, which counters the macaque APOBEC3G restriction. HIVA(Q23)/SIV(vif) replicated poorly in pig-tailed macaque (Ptm) lymphocytes, but viruses were adapted to Ptm lymphocytes. Two independent mutations in gp120, G312V (V3 loop) and A204E (C2 region), were identified that increased peak virus levels by >100-fold. Introduction of G312V and A204E to multiple subtype A Envs and substitution of G312 and A204 with other residues increased entry into Ptm cells by 10- to 100-fold. G312V and A204E Env variants continued to require CCR5 for entry but were up to 50- and 200-fold more sensitive to neutralization by IgG1b12 and soluble CD4 and had a 5- to 50-fold increase in their ability to utilize Ptm CD4 compared to their wild-type counterparts. These findings identify the inefficient use of Ptm CD4 as an unappreciated restriction to subtype A HIV-1 replication in Ptm cells and reveal amino acid changes to gp120 that can overcome this barrier.     

Mechanisms of gentamicin-induced dysfunction of renal cortical mitochondria: II. Effects on mitochondrial monovalent cation transport

Mitochondrial swelling techniques were used to evaluate the effects of the aminoglycoside antibiotic gentamicin on renal cortical mitochondrial monovalent cation permeability. Gentamicin behaved like EDTA to enhance energy-dependent Na⁺- and K⁺-acetate uptake with a relatively greater effect on Na⁺-acetate uptake. Mg²⁺ prevented and reversed the effects of both EDTA and gentamicin. Neither agent affected energy-independent uptake of Na⁺ and K⁺-acetate. Gentamicin did not enhance energy-independent uptake of K⁺- and Na⁺-nitrate. Gentamicin enhanced energy-dependent swelling in a chloride- and phosphate-containing medium as a function of the medium Na⁺ and K⁺ concentration. This effect occurred simultaneously with gentamicin-induced stimulation of State 4 respiration and was blocked by Mg²⁺. Gentamicin did not affect phosphate transport. The results are taken to indicate a specific action of gentamicin to enhance mitochondrial monovalent cation permeability at an Mg²⁺-sensitive site and it is proposed that this accounts for the effects of gentamicin on mitochondrial respiration.     

Interaction between growth factors and retinoic acid in the induction of kidney tubulogenesis in tissue culture.

Kidney tubulogenesis is the initial step in renal organogenesis. The precise molecular determinants of this pattern formation are presently unknown, although soluble factors, such as growth factors, and insoluble factors, such as extracellular matrix molecules, most likely play fundamental roles in this process. To define the molecular determinants of renal proximal tubule morphogenesis, primary cultures of rabbit renal proximal tubule cells in hormonally defined, serum-free media were treated with transforming growth factor-beta 1 (TGF-beta 1), epidermal growth factor (EGF), and the retinoid, all trans-retinoic acid (RA), singly or in combination. Utilizing phase contrast and light and transmission electron microscopy, the simultaneous administration of TGF-beta 1 (10 ng/ml), EGF (1 nM), and RA (0.1 nM) transformed a confluent monolayer of renal proximal tubule cells within 5 to 6 days into three-dimensional cell aggregates containing lumens within the interior of the cell clusters. The lumens were bordered by tubule cells possessing a polarized epithelial cell phenotype with extensive microvilli formation and tight junctional complexes along the luminal border. All three factors were necessary and sufficient to induce this phenotypic transformation. Further studies demonstrated that RA promoted the deposition of the A and B1 chains of laminin, a cell attachment protein of the basement membrane, in a small subset of proximal tubule cells in culture, as deduced by indirect immunofluorescent microscopy. Additional studies demonstrated that soluble purified laminin fully substituted for RA in this system to promote renal tubulogenesis when combined with TGF-beta 1 and EGF. These results demonstrate that the growth factors, TGF-beta 1 and EGF, and the retinoid, RA, promote tubulogenesis in adult renal proximal tubule cells in tissue culture in a manner reminiscent of inductive embryonic kidney morphogenesis. These observations define a coordinated interplay between growth factors and retinoids to induce pattern formation and morphogenesis. Furthermore, the demonstration of RA-induced laminin deposition as a critical event in this morphogenic process identifies laminin as a possible target protein for RA to act as a morphogen.     

Two siphonostomatoid copepods (Coralliomyzontidae) associated with the ahermatypic coral Tubastraea in the Moluccas

Two siphonostomatoid copepods (Coralliomyzontidae) are associatedwith the ahermatypic coral Tubastraea micracantha at Banda inthe Moluccas. Cholomyzon brevisetigerum, new species, hasunusually short setae on the caudal rami. Corallimyzonlatitergum, new species, has an enlarged, posteriorly slightlytrilobate tergum on the third metasomal somite and lacksleg 4.