Mucosal surfaces of the reproductive tract as well as their secretions have important roles in preventing sexual transmission of HIV-1. In the current study, the majority of the intrinsic anti-HIV-1 activity of human seminal plasma (SP) was determined to reside in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV-1 infection, even when SP was diluted 3200-fold. Subsequent fractionation by continuous flow acid-urea (AU)-PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti-HIV-1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti-HIV-1 activity of SP. One peptide fragment of semenogelin I, termed SG-1, was purified from SP by a multistep chromatographic approach, protein sequenced, and determined to exhibit anti-HIV-1 activity against HIV-1. Anti-HIV-1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti-HIV-1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV-1, and that semenogelin-derived peptides contribute to the intrinsic anti-HIV-1 activity of SP.

We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called “SEVI” and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1∶200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.

Porcine granulosa cells isolated from small (1-3 mm in diameter) follicles proliferate rapidly in culture in response to 10% fetal bovine serum (FBS) and epidermal growth factor (EGF) (10 ng/ml). Transforming growth factor-beta (TGF beta) inhibits FBS/EGF-stimulated proliferation in a dose-dependent manner. We have used this proliferation inhibitory property of TGF beta to assay qualitatively, the presence of this growth factor in conditioned medium from cultured follicle cells as well as in partially purified preparations from porcine ovarian compartments. In addition, the concentration of TGF beta in the theca cell conditioned medium was quantitatively estimated by generating a TGF beta-dose-response curve (inhibition of FBS/EGF-stimulated proliferation of granulosa cells in monolayer culture) using authentic human TGF beta-1. Ovarian thecal cells isolated from small and large size follicles in the pig ovary secrete TGF beta-like activity in vitro. Medium conditioned by thecal cells in primary monolayer culture contains a latent form of TGF beta which can be activated by heat or acid treatment. In contrast, and unlike rat granulosa cells, porcine granulosa cells in primary monolayer culture do not secrete detectable levels of TGF beta-like activity in the medium. Incubation of heat-activated thecal cell conditioned medium with a TGF beta-neutralizing antibody (which recognizes TGF beta-1 and 2) but not nonimmune serum attenuated the TGF beta-like activity in thecal cell conditioned medium suggesting that this activity is due to authentic TGF beta. Since many cell types secrete latent TGF beta in the medium when cultured in vitro, we next investigated whether thecal cell secretion of latent TGF beta was a function of cell culture or whether the ovarian thecal compartment actually contained detectable levels of TGF beta-like activity. To this end, we used an acid-ethanol extraction procedure to isolate thecal proteins from fresh-frozen tissue. The acid-ethanol extracted protein fraction was mixed with trace amounts of 125I-TGF beta for detection and chromatographed on Bio-Gel P-60 column under acidic conditions. Elution of TGF beta bioactivity from the Bio-Gel P-60 column as measured by inhibition of granulosa cell proliferation correlated with the elution of radioiodinated authentic TGF beta. Preincubation of TGF beta-like activity-containing fractions with TGF beta-neutralizing antibody attenuated the proliferation-inhibitory activity in these fractions. TGF beta activity was also observed in fractions extracted from porcine corpora lutea.(ABSTRACT TRUNCATED AT 400 WORDS)

An important but poorly understood aspect of mammalian follicle development involves the regulation of theca cell proliferation. To investigate the premise that growth factors regulate theca cell proliferation, porcine theca cells were prepared by collagenase/DN'ase digestion of follicle linings after the removal of the granulosa cells and allowed to attach for 24 h. This method provided a monolayer of theca cells that had little if any granulosa cell contamination and which secreted high levels of androstenedione relative to granulosa cells during moderate-term culture (33-fold difference, P less than 0.01). In medium containing fetal calf serum (10%), theca cells were significantly more responsive to platelet-derived growth factor (PDGF) than epidermal growth factor (EGF) in terms of proliferation (13.4 +/- 0.2-vs. 7.0 +/- 0.1-fold increases relative to the initial cell count, P less than 0.05). This is in contrast to granulosa cells which were significantly more responsive to EGF than PDGF (7.1 +/- 0.1 vs. 4.0 +/- 0.2 fold-increases, P less than 0.05). Since serum has been shown to contain both EGF and PDGF, proliferation studies were performed using plasma-derived serum (PDS) which is growth factor restricted to examine more closely the direct effects of growth factors. In medium containing 0.25% PDS and within experiments, PDGF (1-25 ng/ml) stimulated theca cell proliferation in a dose-dependent manner (2.3-fold increase relative to controls, P less than 0.05) whereas EGF did not. EGF, however, markedly enhanced the proliferative action of PDGF (6.4-fold increase relative to controls, P less than 0.05). Insulin-like growth factor I and low density lipoprotein, factors which enhance markedly the proliferative effects of EGF and PDGF in terms of granulosa cell proliferation, exhibited only a modest synergistic effect with respect to EGF and PDGF upon theca cells (9.5-fold increase vs. a 6.4-fold increase above controls, P less than 0.05). Temporal studies in vitro indicate that theca cell proliferation is low during the first 3-day exposure to growth factors irrespective of treatment (a 2-fold increase over the seeding density). During the second 3-day exposure, however, theca cell proliferation increases 4- to 5-fold. The temporal pattern of theca cell proliferation stimulated by fetal calf serum supplemented with EGF or PDGF and PDS-containing medium supplemented with PDGF, EGF, insulin-like growth factor I, and low density lipoprotein is similar. These results suggest that PDGF is a major mitogen toward porcine theca cells and that EGF greatly enhances its activity.(ABSTRACT TRUNCATED AT 400 WORDS)

The introduction of the technique of intracytoplasmic sperm injection to achieve fertilization, especially using surgically retrieved testicular or epididymal sperm from men with obstructive or non-obstructive azoospermia, has revolutionized the field of assisted reproduction. The techniques for the retrieval of spermatozoa vary from relatively simple percutaneous sperm aspiration to open excision (testicular biopsy) and the more invasive Micro-TESE. The probability of retrieving spermatozoa can be as high as 100% in men with obstructive azoospermia (congenital bilateral absence of the vas deferens, status post-vasectomy). However, in non-obstructive azoospermia, successful sperm retrieval has been reported in 10-100% of cases by various investigators. The surgical retrieval and cryopreservation of sperm, especially in men with non-obstructive azoospermia, to some extent ensures the availability of sperm at the time of intracytoplasmic sperm injection. In addition, this strategy can avoid unnecessary ovarian stimulation in those patients intending to undergo in vitro fertilization-intracytoplasmic sperm injection with freshly retrieved testicular sperm when an absolute absence of sperm in the testis is identified. Several different methods for the cryopreservation of testicular and epididymal sperm are available. The choice of the container or carrier may be an important consideration and should take into account the number or concentration of the sperm in the final preparation. When the number of sperm in a testicular biopsy sample is extremely low (e.g., 1-20 total sperm available), the use of an evacuated zona pellucida to store the cryopreserved sperm has been shown to be an effective approach.