Mónica S. Cameo

Antiacrosin antibodies and infertility. II. Gene immunization with human proacrosin to assess the effect of immunity toward proacrosin/acrosin upon protein activities and animal fertility

OBJECTIVEnTo assess the effect of antiacrosin antibodies upon proacrosin/acrosin activities and animal fertility.nnnDESIGNnProspective study.nnnSETTINGnBasic research laboratory.nnnPATIENT(S)nA gene immunization (GI) model was developed; mice were injected with the sequence encoding human proacrosin (h-proacrosin), cloned in an expression vector.nnnINTERVENTION(S)nSubcloning of h-proacrosin in a eukaryotic expression vector (promoter, CMV; leader sequence, alpha-1 antitrypsin; pSF2-Acro); GI of female mice with this plasmid.nnnMAIN OUTCOME MEASURE(S)nThe following parameters were evaluated: [1] adequate conditions for GI protocols, [2] humoral response to GI with pSF2-Acro, [3] protein regions recognized by the antibodies, and [4] effect of antibodies upon proacrosin/acrosin-ZPA binding and amidase activity, and animal fertility.nnnRESULT(S)nConditions of female mice GI with the proacrosin sequence were established (plasmid purification with anion exchange chromatography and 40 microg of pSF2-Acro per dose) to trigger an immune response, reaching maximum levels at week 9 after the first injection. Antibodies produced by GI recognized human and mouse sperm acrosin systems, inhibited human proacrosin/acrosin interaction with recombinant human ZPA and protease activity, and negatively affected mouse IVF and early embryonic development. In addition, mice immunized with SF2-Acro exhibited a significantly lower size of fetuses.nnnCONCLUSION(S)nAntiacrosin antibodies developed by using GI inhibit human proacrosin/acrosin activities and impair mouse fertility.

The activation of cultured epididymal tubules by androgens

We have studied the influence of androgens, added to the media in which rat epididymal tubules were cultured, upon RNA synthesis. The presence of 1 × 10−5 M dihydrotestosterone (DHT) in the medium during a 3 day culture period induced a 66% increase in the incorporation of 14C-uridine into acid insoluble material. This effect could also be elicited by 5α-androstane-3α, 17β-diol, a potent androgen, but neither by estradiol-17β nor corticosterone or progesterone. Furthermore, it was blocked by the simultaneous presence of cyproterone acetate in the medium, thus stressing the specific nature of the androgenic stimulation. Twelve and 36 h of exposure to DHT (1 × 10−6 M) induced an acceleration in the synthesis of 28S RNA accompanied by minor stimulations of the synthesis of 18S and 4S RNA. Androgens also had a pronounced affect on the aggregation of ribosomes into heavy polyribosomes which showed increased activity in protein synthesis. These results are interpreted as indirect evidence for the synthesis of messenger RNA.

Determination of the binding capacity and affinity of proteins from human pregnancy plasma for androstenediol.

Abstract Proteins from late pregnancy plasma were able to bind androstenediol (3β,17β-dihydroxyandrost-5-ene). This steroid and testosterone compete for the binding sites, displacing each other with apparently similar affinity. Estradiol-17β had only 89% of the displacing effect of equal amounts of cold androstenediol. 60% of the plasma binding activity was lost after heating at 60°C for 30′. The binding capacity of pregnancy plasma for androstenediol was: 0.93 ug/g of protein, with apparently two different binding components in the system. A purified globulin fraction had a capacity of 1.35 ug/g of protein. The combining affinity for 1% plasma at 4°C was: 3.49 × 10−3 ml/ug± 0.48 × 10−3. For the globulin fraction the value was: 9.25 × 10−3 ml/ug ± 0.31 × 10−3. The evidence suggests that androstenediol is bound by the same protein already described as testosterone and estradiol binding globulin.

Further characterization of a model system for the study of human epididymal physiology and its relation to sperm maturation.

Some preliminary speculations about the possible participation of epididymal antigens in sperm function may be supported by the above data. On the one hand, the reduction in the amount of antigens and their abnormal localization on spermatozoa from infertile patients may be coincident with our view about participation of epididymal antigens in the development of zona pellucida binding ability and fertilizing capacity by spermatozoa during maturation. This hypothesis is derived from experiments showing that immature hamster spermatozoa gain their ability to recognize and bind to zona pellucida and to penetrate homologous oocytes when exposed to preparations enriched in androgen-dependent epididymal secretory proteins or preincubated in conditions that favor their interaction with these proteins. Supporting our viewpoint for such a role in humans is evidence showing the progressive development of the ability to interact with hamster denuded oocytes as human spermatozoa pass along the epididymis. On the other hand, the apparent correlation between the loss of epididymal antigens during capacitation and the increased fertilization of human oocytes in vitro may be reminiscent of the removal of a decapacitation or acrosome stabilizing factor known to occur in many species and that must be removed prior to fertilization. Pending further understanding of their physiological role, the androgen-dependent epididymal proteins may become a useful marker of epididymal function and/or of sperm capacitation in humans. Within this context, we wish to stress the potential value of the model system that we have developed for the study of human epididymal physiology.

Abnormal distribution of epididymal antigens on spermatozoa from infertile men**Supported in part by the National Research Council of Argentina, Serono Laboratories, Inc., Randolph, Massachusetts, and grant 15920 from the National Institute of Child Health and Human Development (to J. B.).

An antiserum raised against human epididymal proteins associated with ejaculated sperm was used to test the hypothesis that the amount and/or localization of these antigens may be altered in men with infertility. With the use of immunofluorescence we found that in sperm from fertile donors 88.4% of the cells had the antigens localized over the acrosomal cap only and 1.3% had most of the antigens at extraacrosomal sites. Fifteen of the 26 infertile men (P1) studied had a similar relative distribution of antigens, but the remaining 11 patients (P2) had a 38-fold increase in cells with extraacrosomal localization of the antigens (40%, P less than 0.005). Using flow cytometry to quantitate immunofluorescence, content of antigen on sperm from patients from population P1 (680 +/- 60 V X 10(-4)) was not different from that of control (835 +/- 53 V X 10(-4], whereas it was significantly lower in sperm from patients from population P2 (554 +/- 64 V X 10(-4), P less than 0.005). Differences could not be correlated with parameters measured by routine semen analysis. Our results suggest a possible relationship between the decreased amount of epididymal antigens or their altered localization on sperm and the infertility of patients from population P2.

THE ACTIVATION OF CULTURED EPIDIDYMAL TUBULES BY ANDROGENS

We have studied the influence of androgens, added to the media in which rat epididymal tubules were cultured, upon RNA synthesis. The presence of 1 × 10–5 M dihydrotestosterone (DHT) in the medium during a 3 day culture period induced a 66% increase in the incorporation of 14C-uridine into acid insoluble material. This effect could also be elicited by 5α-androstane-3α, 17β-diol, a potent androgen, but neither by estradiol-17β nor corticosterone or progesterone. Furthermore, it was blocked by the simultaneous presence of cyproterone acetate in the medium, thus stressing the specific nature of the androgenic stimulation. Twelve and 36 h of exposure to DHT (1 × 10–6 M) induced an acceleration in the synthesis of 28S RNA accompanied by minor stimulations of the synthesis of 18S and 4S RNA. Androgens also had a pronounced affect on the aggregation of ribosomes into heavy polyribosomes which showed increased activity in protein synthesis. These results are interpreted as indirect evidence for the synthesis of messenger RNA.