Abstract. Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The rnAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 lxg/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75 %. The average number of sperm fused per egg was also reduced by 75 %. In contrast a control mAb, PH-I, preincubated with sperm at 400 gg/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two ~5I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes.Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears.After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to ,,o49 kD and ,033 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44-and 60-kD bands. In the absence of detergent, the purified protein precipitates, suggesting that either 60 or 44 kD could be an integral membrane polypeptide.

Previous work has indicated that the guinea pig sperm membrane protein, PH-20, functions in sperm-egg adhesion and that its surface expression is regulated by the acrosome reaction. The PH-20 protein was purified by monoclonal antibody affinity chromatography. Sixty-seven to one hundred percent of the PH-20 antigenic activity present in an octylglucoside (OG) extract of sperm was recovered in the purified protein. From 10(10) sperm, approximately 0.4 mg of PH-20 protein was obtained, which was about 0.24% of the total protein in the OG extract. The purified protein retained the ability to bind the three anti-PH-20 monoclonal antibodies we have isolated. Silver staining of purified PH-20 on overloaded sodium dodecyl sulfate (SDS) gels allowed the estimate that silver-stainable contaminants were present at a level of one part in 2000. The purified PH-20 protein exists in three forms separable on SDS-polyacrylamide gel electrophoresis: a major form with a molecular mass of 64 kDa, a minor form of 56 kDa, and an endoproteolytically cleaved form composed of two disulfide-linked fragments of 41-48 kDa and 27 kDa. Cleveland digests of the 64 kDa and 56 kDa polypeptides indicated that they were structurally related. A proportion of the 64 kDa polypeptide in each purified preparation had undergone endoproteolysis at a specific site, so that it was cleaved into the two disulfide-linked fragments, 41-48 kDa and 27 kDa. It is speculated that the site-specific endoproteolysis of PH-20 may occur during the acrosome reaction and have biological significance.

We describe Pichia caribaea, a new species of yeast which is closely related to P. amethionina. P. caribaea, of which 92 strains were isolated, is heterothallic and occurs in nature in both the haploid state and the diploid state. It produces asci with four hat-shaped spores, which are gradually released upon maturity. P. caribaea occurs in rotting tissue of cereoid and opuntia cacti on various islands in the greater Caribbean area and on coastal land masses surrounding it. It resembles P. amethionina var. pachycereana in its assimilation pattern of carbon compounds but differs in its ability to ferment glucose strongly. The DNAs of P. caribaea and the two described varieties of P. amethionina show about 40% complementarity. The type strain of P. caribaea is strain UCD-FST 81-62 (= ATTC 76713 = CBS 7692).In 1978, Starmer et al. (15) described Pichia amethionina, a naturally occurring auxotroph that has an absolute requirement for organic forms of sulfur, such as the sulfur-containing amino acids L-methionine and L-cysteine. This nonfermentative species is represented by two varieties, P. amethionina var. amethionina and P. amethionina var. pachycereana. The former is commonly recovered from rot pockets in cacti of the subtribe Stenocereinae, whereas the latter has its principal habitat in members of the subtribe Pachycereinae (2, 15). All isolates of these two varieties came from decaying columnar and Opuntia cacti in the North American Sonoran desert, including the Baja California peninsula. The DNA relatedness between the two varieties was determined by Holzschu and Phaff (3), who reported a mean DNA-DNA complementarity value of 64.9% 2 3.2% on the basis of 16 pairwise comparisons, including reverse DNA reassociations. Nearly all strains of P. amethionina var. amethionina lack the ability to utilize D-mannitol for growth and are resistant to triterpene glycosides or digitonin at 25"C, while P. amethionina var. pachycereana is D-mannitol positive and is inhibited by these compounds During two expeditions in 1982 and 1983 to collect yeasts from cactus necroses on various islands in the Caribbean Sea and the Bahamas, we discovered another yeast with an absolute requirement for sulfur-containing amino acids that was similar to P. amethionina var. pachycereana, except that glucose was vigorously fermented. Additional strains with these properties had been isolated by J. W. Fell and later by M. A. Lachance in the Exuma chain in the Bahamas, by Starmer and Phaff (14) on the Gulf of Mexico coast of Texas, and more recently by Starmer et al. (10) in Florida. In the publications cited, these isolates were referred to as P. amethionina var. "7' or "fermentans." As a formal description with a Latin diagnosis was not provided, this name constitutes a nomen nudum.To substantiate the basis for a valid description of this group of isolates, we carried out mating tests and DNA-DNA hybridizations between typical strains of the fermenting isolates and the two varieties of P. amethionina. Mating between the fermenting isolates a...

We describe Pichiu barkeri, a new cactophilic yeast species occurring in necrotic tissue of Opuntia stricta in New South Wales, Australia, and on several islands in the Caribbean Sea. The new species is homothallic and appears to occur in nature in the haploid state. After conjugation between a mother cell and a bud, four hat-shaped spores are produced that are rapidly released from the ascus. The range of guanine-plus-cytosine contents for the nuclear deoxyribonucleic acids of 22 strains is 35.7 to 36.6 mol% (average, 36.1 mol%; standard deviation, 0.2 mol%). The type strain of P. barkeri is strain UCD-FST 83-994.3 (ATCC 64111, CBS 7256) isolated at Discovery Bay, Jamaica.The rotting stems and cladodes of Opuntia species are specific habitats for a diversity of cactus-specific yeast species (1,2,7,11). During surveys of yeasts associated with rots of Opuntia species in Australia (1, 2), we isolated a number of strains that were initially identified by the system of Lodder (5) as Pichia kluyveri. Because P. kluyveri had been recovered only rarely in earlier surveys of columnar cacti (12), we initially concluded that these strains might have converged on the P . kluyveri phenotype or might be fermentative forms of Pichia cactophila. Subsequent characterization by molecular criteria (6) showed that the isolates in question differed from P. kluyveri in having a nuclear deoxyribonucleic acid (DNA) guanine-plus-cytosine (G + C) content approximately 6 mol% higher than that of P. kluyveri (-36 versus -30 mol%), virtually precluding conspecificity (6). Ensuing experiments, using the technique of DNA-DNA hybridization, provided additional evidence that the Australian isolates represented a different species, although retaining a distant relationship with P. kluyveri. DNA-DNA hybridization experiments also showed that the Australian isolates are distinct from P. cactophila, which has a similar base composition (-36 mol% G+C). During subsequent explorations of the yeast floras associated with cacti occurring on various islands in the Caribbean Sea, we isolated several additional strains of the new species, Pichia barkeri, also associated with necrotic tissue of Opuntia stricta.