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The effect that organic urinary macromolecules have on the crystallization of calcium oxalate from a synthetic urine-like solution was studied in a mixed suspension-mixed product removal (MSMPR) continuous crystallizer. Precipitation of calcium oxalate crystals occurs during the continuous passage of urine through the renal system (kidney, bladder and tubules). While in normal circumstances these crystals remain small in size and exit the system unimpeded, in the pathologic condition calcium oxalate crystals are observed to aggregate and grow beyond a critical size where there is a significant probability of being trapped inside the renal system, e.g., on the kidney wall or in the tubules. Once trapped, the crystals become a nidus for further solute deposition and aggregation, giving origin to a renal calculus or stone. It is shown that this process is significantly affected by the presence or absence of organic macromolecules that act as modifiers of crystal growth, nucleation, and aggregation. An ultrafiltration technique was used to fractionate urine specimens from normal (N) and stone-forming (SF) persons into organic compounds of different molecular weight. These compounds were then added to the MSMPR system to test their effect on calcium oxalate crystallization. Significant differences were found to exist between N and SF urines in the composition, molecular weight distribution, and total quantity of these organic macromolecular compounds. The fraction of macromolecules responsible for the major effects on calcium oxalate crystallization was isolated, and its effect on crystal growth and nucleation rates was quantified. The steady state driving force (supersaturation) in the MSMPR system was measured. Striking differences in supersaturation versus residence time behavior between N and SF macromolecules were observed. The experimental conditions under which calcium oxalate crystals agglomerate were identified. Evidence which supports agglomeration as a key mechanism in urinary stone formation is presented.

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The developmental potential of mammalian oocytes cryopreserved with procedures similar to those used for embryos has been limited, inasmuch as oocytes differ from embryos in advanced stages of development, both physiologically and morphologically. The objective of this work was to elucidate the precise nature of freeze-thaw damage with the expectation that identification of specific targets will enable devising optimal procedures for cryopreservation of bovine oocytes to prevent specific damage and minimize the loss of developmental capacity. / In the first series of experiments, bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Survival was assessed morphologically and also by in vitro fertilization (IVF) and culture (IVC). Morphological integrity and developmental capacity were greater in S-oocytes than in V-oocytes (P $<$ 0.05). Transfer of four embryos (2 morulae and 2 blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in the birth of twin calves. / In the second series of experiments, oocytes (GV and IVM) were exposed to a cryoprotectant solution (DAP213: 2M DMSO, 1M acetamide, 3M propanediol) for 1.5 or 5 min and viability assessed by IVM-IVF-IVC. Oocytes were also examined by transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound premature cortical granule (CG) release and vesiculation. These changes were less pronounced in oocytes exposed to DAP213 only after IVM. The results suggest that: (1) the extrusion of CG is one of the important cytological events affected by the treatment of oocytes with DAP213; (2) GV oocytes are more sensitive to the cryoprotectant than IVM oocytes. / In the third series of experiments, GV and IVM oocytes were vitrified with DAP213. On rewarming, DAP213 was removed by a one- or three-step dilution procedure and survival assessed by development after IVM-IVF-IVC. Morphology was assessed by TEM study immediately following DAP213 removal. Both assessments indicated that: (1) IVM oocytes are more tolerant to vitrification than are GV oocytes; (2) the three-step dilution is less damaging than the one-step procedure; (3) changes in the zona pellucida (loss of plasticity) of IVM oocytes following vitrification may result from the premature release of cortical granules.