Semen Liquefaction Research Articles

Role of human prostasomes in the activation of spermatozoa.

Prostasomes are small vesicles of prostatic origin contained in human semen. Their composition is peculiar under many aspects. Cholesterol is abundant and many proteins are endowed with enzymatic or other activities. The function of prostasomes has been amply debated and several hypotheses have been put forward. The liquefaction of semen, spermatozoa motility, antibacterial activity and immunological functions have been related to prostasomes. Under certain aspects, prostasomes resemble synaptosomes. The fusion of prostasomes to spermatozoa enriches spermatozoa with cholesterol and causes bursts of cytoplasmic sperm calcium. The interaction of spermatozoa and prostasomes should be limited to vagina since prostasomes are immobile and do not follow spermatozoa in the superior female genital tract. Calcium bursts would increase spermatozoa motility, where cholesterol would decapacitate spermatozoa, so preventing untimely activation. Since spermatozoa receive many different molecules from prostasomes, additional effects are also possible. Prostasomes makes spermatozoa more apt to be activated by progesterone in the proximity of the ovum. Therefore, the fusion between spermatozoa and prostasomes would influence spermatozoa behaviour under many aspects and might be relevant for fecundation. The richness of molecular species in prostasomes is amazing and these small vesicles are expected to lead to many more discoveries in the field of human reproduction.

Semen collection, cryopreservation and artificial insemination in the dromedary camel

Collection of semen with a bovine artificial vagina (AV) was attempted with each of 14 camels over a period of 2 years. Semen samples were evaluated, extended and cryopreserved. Frozen thawed semen, diluted cooled semen or whole semen was used to inseminate some female camels which were induced to ovulate with hCG. Males ejaculated semen into the AV in 74.6% collection attempts. The male copulated for at least 200 s in 62.9% attempts. The remaining copulations were of shorter duration. Similarly, 49.3% ejaculates were at least 3 ml of semen. Libido and donation of semen improved from December onwards and reached a peak after mid January with peak performance persisting until April. It declined during May. The majority of camels had lost libido and refuse to donate semen by the end of May. Camel semen is in gel form. While 35.9% of 203 semen samples exhibited no individual sperm motility, 28.5% exhibited low to fair grade individual sperm motility and only 35.4% exhibited >50% sperm motility. Differences existed between animals ( P<0.01) and months ( P<0.05) of collection, while effect of copulation time was not significant. Mass motility was not observed in camel semen. Individual sperm motility develops after liquefaction of semen. Addition of caffeine but not chymotrypsin improved the individual motility. The mean live percent sperm count and normal acrosome were 73.3±1.0 and 92.0±0.5, respectively. Only 51.1% of 45 semen samples with pre-freeze motility of >50% and 25% of 16 semen samples from low pre-freeze motility group with an overall success of 44.2% of 61 semen samples were successfully preserved. Wide variation was observed in the freezability of semen from different males. Attempts to impregnate female camels with liquid semen, frozen thawed semen and whole semen after hCG induced ovulation resulted in 0/10, 1/13 and 4/10 pregnancies.

The Synthesis of a Prodrug of Doxorubicin Designed to Provide Reduced Systemic Toxicity and Greater Target Efficacy

Doxorubicin (Dox) can provide some stabilization in prostate cancer; however, its use is limited because of systemic toxicities, primarily cardiotoxicity and immunosuppression. The administration of a prodrug of doxorubicin, designed to permit selective activation by the tumor, would reduce general systemic exposure to the active drug and would thereby increase the therapeutic index. Prostate specific antigen (PSA) is a serine protease with chymotrypsin-like activity that is a member of the kallikrein gene family. PSA's putative physiological role is the liquefaction of semen by virtue of its ability to cleave the seminal fluid proteins semenogelins I and II. Serum PSA levels have been found to correlate well with the number of malignant prostate cells. The use of a prodrug which is cleaved by the enzyme PSA in the prostate should in principle produce high localized concentrations of the cytotoxic agent at the tumor site while limiting systemic exposure to the active drug. Cleavage maps following PSA treatment of human semenogelin were constructed. Systematic modification of the amino acid residues flanking the primary cleavage site led to the synthesis of a series of short peptides which were efficiently hydrolyzed by PSA. Subsequent coupling of selected peptides to doxorubicin provided a series of doxorubicin-peptide conjugates which were evaluated in vitro and in vivo as targeted prodrugs for PSA-secreting tumor cells. From these studies we selected Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox, 27, as the peptide-doxorubicin conjugate with the best profile of physical and biological properties. Compound 27 has a greater than 20-fold selectivity against human prostate PSA-secreting LNCaP cells relative to the non-PSA-secreting DuPRO cell line. In nude mouse xenograft studies, 27 reduced PSA levels by 95% and tumor weight by 87% at a dose below its MTD. Both doxorubicin and Leu-Dox (13) were ineffective in reducing circulating PSA and tumor burden at their maximum tolerated doses. On the basis of these results, we selected 27 for further study to assess its ability to inhibit human prostate cancer cell growth and tumorigenesis.

Effect of Cr(VI) Exposure on Sperm Quality: Human and Animal Studies

The semen status of male workers occupationally exposed to hexavalent chromium(VI) was investigated. Sperm counts from exposed workers were 47.05+/-2.13 x 10(6)/ml and those from control group 88.96+/-3.40 x 10(6)/ml. Sperm motility decreased from 81.92+/-0.41% for the control group to 69.71+/-0.93% for the exposed workers. The levels of zinc, lactate dehydrogenase (LDH), and lactate dehydrogenase C4 isoenzyme (LDH-x) in seminal plasma for the exposed workers were 1.48+/-0.07 micromol/ml, 1.05+/-0.02 x 10(3) U, and 0.47+/-0.01 x 10(3) U, respectively, which were significantly lower than those of 5.72+/-0.15 micromol/ml, 1.49+/-0.02 x 10(3) U, and 0.78+/-0.15 x 10(3) U for the control group, respectively. Follicle stimulating hormone (FSH) (7.34+/-0.34 x 10(-3) IU/ml) in serum from the exposed workers was significantly higher than that (2.41+/-0.08 x 10(-3) IU/ml) from the control group. On the other hand, there were no significant differences in semen volume, semen liquefaction time, luteinizing hormone (LH) level in serum, and Cr concentration in both serum and seminal plasma between the exposed workers and the control group. Feeding Cr(VI) to rats significantly reduced the epididymal sperm counts from 87.40+/-3.85 x 10(6)/g epididymis in control group to 21.40+/-1.20 x 10(6)/g epididymis at a CrO(3) dose of 10 mg/kg body weight and to 17.48+/-1.04 x 10(6)/g epididymis at a CrO(3) dose of 20 mg/kg body weight. Exposure of rats to Cr(VI) also significantly increased the sperm abnormality from 2.75+/-0.06% in the control group to 6.68+/-0.32% in the exposed group at a CrO(3) dose of 10 mg/kg body and to 7.6+/-0.15% at a CrO(3) dose of 20 mg/kg body weight. In exposed rats, there was visible disruption in germ cell arrangement near the walls of the seminiferous tubules. The diameters of seminiferous tubules in exposed rats were smaller. These results suggest that occupational exposure to chromium(VI) leads to alteration of semen status and may affect the reproductive success of exposed workers.

Effect of Cr(VI) Exposure on Sperm Quality: Human and Animal Studies

The semen status of male workers occupationally exposed to hexavalent chromium(VI) was investigated. Sperm counts from exposed workers were 47.05±2.13×106/ml and those from control group 88.96±3.40×106/ml. Sperm motility decreased from 81.92±0.41% for the control group to 69.71±0.93% for the exposed workers. The levels of zinc, lactate dehydrogenase (LDH), and lactate dehydrogenase C4 isoenzyme (LDH-x) in seminal plasma for the exposed workers were 1.48±0.07 μmol/ml, 1.05±0.02×103 U, and 0.47±0.01×103 U, respectively, which were significantly lower than those of 5.72±0.15 μmol/ml, 1.49±0.02×103 U, and 0.78±0.15×103 U for the control group, respectively. Follicle stimulating hormone (FSH) (7.34±0.34×10−3 IU/ml) in serum from the exposed workers was significantly higher than that (2.41±0.08×10−3 IU/ml) from the control group. On the other hand, there were no significant differences in semen volume, semen liquefaction time, luteinizing hormone (LH) level in serum, and Cr concentration in both serum and seminal plasma between the exposed workers and the control group. Feeding Cr(VI) to rats significantly reduced the epididymal sperm counts from 87.40±3.85×106/g epididymis in control group to 21.40±1.20×106/g epididymis at a CrO3 dose of 10 mg/kg body weight and to 17.48±1.04×106/g epididymis at a CrO3 dose of 20 mg/kg body weight. Exposure of rats to Cr(VI) also significantly increased the sperm abnormality from 2.75±0.06% in the control group to 6.68±0.32% in the exposed group at a CrO3 dose of 10 mg/kg body and to 7.6±0.15% at a CrO3 dose of 20 mg/kg body weight. In exposed rats, there was visible disruption in germ cell arrangement near the walls of the seminiferous tubules. The diameters of seminiferous tubules in exposed rats were smaller. These results suggest that occupational exposure to chromium(VI) leads to alteration of semen status and may affect the reproductive success of exposed workers.

Chromatin decondensation of human sperm in vitro and its relation to fertilization rate after ICSI.

The inability of sperm chromatin to decondense has been implicated in the failure of fertilization, This study was undertaken to identify the relationship between sperm chromatin decondensation in vitro after incubation with follicular fluid at various points in time and fertilization or pregnancy rates after intracytoplasmic sperm injection. Moreover, an attempt was made to determine whether this test could be used as a predictive test for the outcome of ICSI. Thirty-two infertile couples undergoing ICSI therapy were included in this prospective study. One milliter of semen from each sample was mixed with 1 mL of follicular fluid obtained from ICSI patients at the time of oocyte retrieval and incubated for 24 h. Many smears were made directly after semen liquefaction at the following time intervals: 30, 60, and 120 min and 24 h. Chromatin decondensation was evaluated with acridine orange staining. The mean percentage of uncondensed chromatin of spermatozoa in the native semen samples was 25 +/- 18.3%, which increased within 24 h to 91 +/- 9.5%. On the other hand, the fertilization and ongoing pregnancy rates were 64 +/- 21.7% and 20%, respectively. However, no correlations were found between chromatin decondensation at various point of time (30, 60, and 120 min and 24 h) and fertilization rate. No correlation was shown between the chromatin decondensation and sperm counts in the ejaculate. morphology, or the percentage of condensed chromatin. In light of this study, chromatin decondensation in vitro cannot be recommended for predicting the fertilization potential of spermatozoa and pregnancy rates in the ICSI program. Further research is necessary, especially in cases where ICSI is being considered as a therapeutic option.

Use of the Multi-ZSC one-step standardized swim-up method: recovery of high-quality spermatozoa for intrauterine insemination or other forms of assisted reproductive technologies

Use of the Multi-ZSC one-step standardized swim-up method: recovery of high-quality spermatozoa for intrauterine insemination or other forms of assisted reproductive technologies

Prostate-specific antigen and new related markers for prostate cancer.

Although prostate-specific antigen (PSA), or human kallikrein 3, is the most valuable tool available for the diagnosis and management of prostate cancer, as currently used it is insufficiently sensitive and specific for early detection or staging of the malignancy. Many new concepts have been introduced in order to optimize the clinical use of PSA measurements, but each one has its own drawbacks. The molecular forms of PSA, especially the free PSA, seem to be useful for the detection of prostate cancer in men with PSA concentrations falling in the 4-10 microg/l range. New molecular techniques, such as reverse transcriptase polymerase chain reaction for the detection of minimal amounts of PSA messenger RNA and prostate-specific membrane antigen, offer new promise for the prognosis and possibly staging of prostate cancer. On the other hand, human kallikrein 2, a serine protease closely related to PSA that is also expressed predominantly in the prostate, may be a new adjuvant marker for prostate cancer. As for its biological functions, PSA can no longer be regarded as a specific prostate molecule associated mainly with semen liquefaction when it has a possible role as a prognostic indicator in female breast cancer. The biological role of PSA in normal tissues and tumors may be much more complex than previously thought and requires further investigation.

Recent advances in andrology research: physiopathology and clinical application to fertility and infertility.

Sperm maturation depends on androgen and is mediated by several unidentified epididymal factors: glycoproteins and metabolites affecting acrosomal stability and fertilizing potential of capacitated sperm. Several genes encoding human epididymis-specific proteins have been described. One of the cloned epididymal cDNA encodes a polypeptide designated as HE4 with an estimated molecular mass of 10,000. Leydig cells are rich in lipid droplets and display epithelioid features. These cells have a cord-like arrangement; the cords are formed by one or two closely apposed cells. In between these cells, labyrinthine or canalicular-like spaces are opened in wide perivascular spaces that improve cell secretion of hormones and facilitate their transport into the blood, as well as the traffic of fluids and metabolites. Coagulation and liquefaction in human semen plays an important role in the capacitation of semen. The liquefaction of semen is retarded by the powerful synthetic inhibitors of 6-amidino-2-naphtyl-p-guanidinobenzoate dimethansulfonate.

The role of prostate-specific antigen in the chemoprevention of prostate cancer

An understanding of the natural history of changes in prostate-specific antigen (PSA) may be valuable as a surrogate view of prostate dynamics, as a method to differentiate between benign and malignant growth, and as a means to assess the use of PSA as a tool for monitoring activity of chemoprevention agents. Although PSA appears to be useful as a noninvasive marker of prostatic growth, PSA changes should not be confused with a direct measure of tumor growth. Serum PSA levels are a function of tumor volume but are also influenced by the volume of benign epithelium, grade of carcinoma (if any), inflammation, androgen levels, growth factors, and the extracellular matrix. The biological functions of PSA in the prostate and in its secretions need to be more completely elucidated in order that PSA measurements may more accurately describe prostate dynamics. The expression of PSA is androgen-regulated. It is one of the most abundant prostate-derived proteins in the seminal fluid. Seminogelin, a major protein in seminal fluid, is cleaved by PSA, and this cleavage is important in the liquefaction of semen. Less is known about other PSA substrates. Current PSA studies indicate that cancer cases exhibit an early slow linear PSA phase followed by a rapid exponential phase, and that PSA levels begin to increase exponentially approximately 7-9 years before diagnosis. The establishment of age-specific PSA reference ranges (ASRR) and of PSA velocity (PSAV) rates provide elements of a baseline from which prediction models could measure malignant potential of a prostatic carcinoma. Moreover, recent discoveries of different molecular forms of PSA in serum may allow a much more accurate differentiation of benign and malignant growth as well as a more potent measure of the impact of chemoprevention agents. If PSA doubling time is approximately 2.4-3.0 years and accurately reflects tumor doubling time, and if the average man has less than 0.5 ml of latent prostatic tumor tissue and the average stage T2 cancer is approximately 4 ml when detected, then the available PSA data suggest that the 3 doublings necessary to change from 0.5-4.0 ml, would take 7-12 years for a typical small volume tumor to reach the size of most stage T2 tumors. The findings that histologic cancers appear at much younger ages than previously known is disturbing. It indicates that disease initiation may begin sooner than ever thought likely. "Normal" PSA levels for younger men (< 40 years of age) may need to be studied, and an emphasis upon premalignant lesions in this age group may be necessary. Younger men may represent the most appropriate population and premalignant lesions the most relevant clinical factor for prostate cancer chemoprevention studies and trials. The molecular composition and molecular changes of PSA derived from premalignant lesions have yet to be elucidated, but such investigations may lead to a more complete understanding of the possible progression or transformation of normal prostate cells to premalignancy and subsequently to carcinoma. High grade prostatic intraepithelial neoplasia (PIN) in and of itself does not account for elevated serum PSA levels, but subtle changes in the molecular dynamics of PSA may reveal the influence of androgens and the impact of chemopreventive agents.

Biochemical characteristics and recent biological knowledge on prostate-specific antigen.

Since its identification in seminal fluid in 1971, much new information has been obtained about the biology and expression of prostate-specific antigen (PSA). PSA is a glycoprotein composed of 93% amino acids and 7% carbohydrates, with a molecular weight of about 30,000 Da. Functionally and structurally PSA is a kallikrein-like serine protease, and its physiologic role is degradation of the major proteins of seminal coagulum (semenogelin I and II, fibronectin), which leads to semen liquefaction. The PSA gene is located on the 13q region of chromosome 19, and it has a high degree of homology (more than 80%) with genes of the human glandular kallikrein (hKGK1). PSA production and expression are preferentially but not exclusively associated to the normal, benign hyperplastic and cancerous tissues of the prostate. In fact, it has been demonstrated that PSA is also present in accessory male sex glands and breast cancer. It was recently reported that PSA was also present in milk of lactating women. Many factors may influence PSA synthesis and production, and among them the most important are androgen, retinoic acid and growth factor stimulation. Significant advances have been recently made as regards the molecular isoforms of PSA. In the seminal fluid PSA seems partially bound to a serpine (protein C inhibitor), whereas in serum it is predominantly associated to alpha-1-antichymotrypsin and in a small quantity to alpha-2-macroglobulin. These new findings will have implications for the clinical application of PSA as a tumor marker for prostate cancer.

Sperm motility inhibitor from human seminal plasma: association with semen coagulum.

Human seminal plasma contains a sperm motility inhibitor (SPMI) originating from the seminal vesicles as a 52 kDa precursor form that is rapidly degraded by prostatic proteases after ejaculation. In this study, the distribution of SPMI biological activity and antigens was analysed in chemically induced, as well as naturally occurring, arrest of semen liquefaction. SPMI activity was detected exclusively in the coagulated semen fraction at 2200 +/- 560 IU, whereas total seminal plasma proteins separated more evenly between soluble and coagulated components (91 +/- 19 and 65 +/- 18 mg, respectively). An SPMI antiserum recognized different forms of SPMI precursors at 52, 38, 35, 33 and 20 kDa in the coagulum while the soluble protein fraction contained only one major immunoreactive band at 15 kDa. High levels of SPMI activity (1500 +/- 180 IU/ml) together with high molecular mass forms of SPMI precursor and low sperm motility (26%) were detected in semen samples that failed to liquefy spontaneously at room temperature. Addition of prostatic secretions to the non-liquefying samples caused a decrease of SPMI activity (330 +/- 17 IU/ml) and transformed the SPMI precursor into low molecular mass forms (14-22 kDa) with a concomitant increase in sperm motility to 49%. The results suggest that SPMI is highly associated with the seminal coagulum components as very active forms that may adversely affect sperm motility when not properly processed after ejaculation.

Biochemical characteristics and biological profile of prostate specific antigen (PSA)

Since its identification in seminai fluid in 1971, a tot more knowledge has been obtained about the biology and expression of prostate specific antigen (PSA). PSA is a glycoprotein consisting of 93% aminoacids and 7% carbohydrates with a molecular weight of about 30000 dalton. Functionally and structurally PSA is a kallikrein-like serine protease and its physiological role is the degradation of the major proteins of the seminal coagulum (semenogelin I and II, fibronectin) leading to semen liquefaction. The PSA gene is located on the 13q region of chromosome 19 and has a high degree of homology (more than 80%) with the gene of human glandular kallikrein (hGK1). PSA production and expression are preferentially but not exclusively associated with the normal, benign hyperplastic and cancerous tissues of the prostate. In fact, it has been demonstrated that PSA is also present in the accessory male sex glands of Cowper, Littre and Morgagni and other extra-prostatic neoplasms, such as salivary gland tumours and breast cancer. Many factors may influence PSA synthesis and production and among them the most important are androgen and growth factor stimulation. Advances have recently been made on the molecular isoforms of PSA. In the seminai fluid PSA seems partially bound to a serpine (protein C inhibitor), whilst in the serum PSA is predominantly associated to α-antichymotrypsin (ACT) and in low quantity to α2-macroglobulin (α2M). All these findings will have implications on clinical applications of PSA as a tumour marker for prostate cancer.

Sperm motility inhibitor from human seminal plasma: association with semen coagulum

Human seminal plasma contains a sperm motility inhibitor (SPMI) originating from the seminal vesicles as a 52 kDa precursor form that is rapidly degraded by prostatic proteases after ejaculation. In this study, the distribution of SPMI biological activity and antigens was analysed in chemically induced, as well as naturally occurring, arrest of semen liquefaction. SPMI activity was detected exclusively in the coagulated semen fraction at 2200 ± 560 IU, whereas total seminal plasma proteins separated more evenly between soluble and coagulated components (91 ± 19 and 65 ± 18 mg, respectively). An SPMI antiserum recognized different forms of SPMI precursors at 52, 38, 35, 33 and 20 kDa in the coagulum while the soluble protein fraction contained only one major immunoreactive band at 15 kDa. High levels of SPMI activity (1500 ± 180 IU/ml) together with high molecular mass forms of SPMI precursor and low sperm motility (26%) were detected in semen samples that failed to liquefy spontaneously at room temperature. Addition of prostatic secretions to the non-liquefying samples caused a decrease of SPMI activity (330 ± 17 IU/ml) and transformed the SPMI precursor into low molecular mass forms (14–22 kDa) with a concomitant increase in sperm motility to 49%. The results suggest that SPMI is highly associated with the seminal coagulum components as very active forms that may adversely affect sperm motilrty when not properly processed after ejaculation.

Dithiothreitol effects on the viscosity and quality of human semen

To determine the effects of treatment with dithiothreitol (DTT) on seminal viscosity, consistency, and sperm motility, motion characteristics, and morphology. Prospective, in vitro study. University teaching hospital. Semen donors and patients of the infertility program. Experiment 1, n = 34; experiment 2, n = 82. Sufficient seminal volume for use was the only selection criterion. Experiment 1: semen was combined with 5.9 mM DTT solution 1:1 (vol/vol), combined with distilled water 1:1 (vol/vol), or untreated. Experiment 2: semen was combined with 5.9 mM DTT solution 1:1 (vol/vol) or untreated. Seminal consistency and viscosity, percentage of motile sperm, sperm motion characteristics (straight line velocity, curvilinear velocity, mean linearity, and angle of lateral head displacement), sperm concentration, and sperm morphology. Treatment of semen with DTT reduced seminal consistency and viscosity. Treatment with DTT had no effect on sperm concentration or percentage motile sperm. Sperm velocity was reduced by DTT treatment, particularly in semen that had normal initial consistency. Morphology of sperm in semen exhibiting abnormal initial consistency was unaffected by DTT. Dithiothreitol effectively induced liquefaction of nonliquefied semen in vitro and had minimal effects on the sperm motility, motion characteristics, and morphology. Evaluation of DTT effects on the chromatin and on other functional traits of human sperm must be conducted before its use can be advocated.

Dihydrotestosterone regulation of semen in male pseudohermaphrodites with 5 alpha-reductase-2 deficiency.

Semen analyses were performed in nine male pseudohermaphrodites with inherited 5 alpha-reductase-2 deficiency and decreased dihydrotestosterone (DHT) production. The semen samples were characterized by extremely low volume (range, < 0.05 to 1.0 mL), increased viscosity, and poor liquefaction. Surgical correction of pseudovaginal perineoscrotal hypospadias in four subjects did not result in an increase in semen volume or a change in viscosity. Inexplicably, semen liquefaction reverted to normal. Affected males have rudimentary prostates and small seminal vesicles. Six subjects had bilaterally descended testes, one subject had bilaterally retractile testes, and two subjects had unilaterally undescended testes. Semen from one subject with bilaterally descended testes had a normal sperm concentration, normal total sperm count, and normal motility and morphology. Semen from another subject who was oligospermic at baseline demonstrated a normal sperm concentration after hypospadias repair, with a low total sperm count. The other subjects studied were oligospermic or azospermic. In summary, DHt appears to regulate semen volume and viscosity through its action on the development and function of the prostate and seminal vesicles. The finding of normal sperm concentrations in two subjects with 5 alpha-reductase-2 deficiency suggests that DHT does not play a major role in spermatogenesis. However, the possibility that low levels of DHT might be sufficient for normal spermatogenesis must also be considered.

The effect of some proteinase inhibitors on liquefaction of human semen.

This study examined the effect of some proteinase inhibitors on liquefaction of human semen. It revealed that a strong plasmin inhibitor, 6-amidino-2-naphthyl-6-guanidinobenzoate dihydrochloride (Fusan) showed a significant inhibition of liquefaction, while t-amino caproic acid (t-ACA) showed a weak retardation effect. In terms of sperm quality after liquefaction, Fusan (10 mM), ethyl diamine tetra-acetic acid (EDTA) and Urinastatin completely inhibited sperm motility. Fusan (1 mM) and Lima bean trypsin inhibitor (LBTI) decreased sperm motility significantly, while leupeptin and t-ACA had little effect. Leupeptin, LBTI, t-ACA and Fusan (1 mM) did not affect sperm speed. 50% inhibition of sperm motility was calculated to be approximately 1.7 mM of Fusan concentration. In addition, two inhibitors, Chymostatin and Phosphoramidon were also tested with each experiment and had no effect on liquefaction or on sperm motility and speed. These results strongly suggest that plasmin may play an important role in the liquefaction process of human semen.

Physical and biochemical characteristics of alpaca semen

Semen was collected from ten intact male alpacas (five were adults and five were 3 years old) using an artificial vagina. Liquefaction of the viscous semen occurred within 24 h, after which samples were centrifuged at 3000 rev min −1 for 20 min before biochemical analysis. The average ejaculate volume was 1.7 ml; it was milky white in color, highly viscous and had slow mass motility. There were no significant differences in the concentrations of chloride, calcium, inorganic phosphate, phospholipids, total nitrogen, total protein and albumin between ages ( P>0.05), with the exception of glucose and globulins concentrations. The mean concentrations of the components were chloride 376.0 mequiv 1 −1, calcium 17.9 mg dl −1, inorganic phosphate 9.6 mg dl −1; glucose 6.2 mg dl −1, phospholipids 28.7 mg dl −1, total nitrogen 597.4 mg dl −1, total protein 3.7 mg dl −1, albumin 2.1 mg dl −1 and globulins 1.6 g dl −1, which is in close agreement with results for other species.

Demonstration of the Role of Prostate‐Specific Antigen in Semen Liquefaction by Two‐Dimensional Electrophoresis

Two-dimensional protein profiles of human semen, prostatic fluid, and seminal vesicle fluid were compared to demonstrate changes in the protein composition of human semen before and after liquefaction. Semen specimens were obtained from a volunteer. Prostatic fluid specimens were collected by rectal massage from patients visiting a urology clinic. Samples of seminal vesicle fluid were collected by needle aspiration from isolated seminal vesicles, which were removed at surgery. All specimens were prepared and processed according to the ISO-DALT system for separation of proteins in two-dimensional gels. Following electrophoresis, protein spots in the gels were visualized by silver staining. Prostatic fluid and seminal vesicle fluid showed their characteristic protein profiles. The protein profile of human semen contained specific proteins of both prostatic fluid and seminal vesicle fluid. One major group of proteins in seminal vesicle fluid (Mw 28,000-68,000 daltons), designated as seminal vesicle-specific antigen, was observed in freshly ejaculated human semen, but disappeared from the two-dimensional profile when the ejaculate was allowed to stand at room temperature for 30 min. When prostatic fluid or prostate-specific antigen was mixed with seminal vesicle fluid and incubated at 37C for 30 min, the seminal vesicle-specific antigen also disappeared from the two-dimensional profiles. The findings indicate that seminal vesicle-specific antigen, a group of predominant proteins in seminal vesicle fluid, is the structural component of seminal coagulum, and that prostate-specific antigen is the enzyme which digests seminal vesicle-specific antigen and liquifies semen coagulum.

Pregnancy rate is significantly higher in in vitro fertilization procedure with spermatozoa isolated from nonliquefying semen in which liquefaction is induced by α-amylase

In 115 patients (group I) in which normal liquefaction of semen occurred, the fertilization rate of the yielded oocytes was 71.2% after standard in vitro fertilization procedure, and the viable pregnancy rate per patient per cycle was 13.0%. In a comparable group of 26 patients (group II), in which semen liquefaction did not occur spontaneously, alpha-amylase was added in order to liquefy the semen. In this group, the fertilization rate was 86.7% of the yielded oocytes, and the viable pregnancy rate per patient per cycle was 26.9% (P less than 0.05). It is concluded that alpha-amylase can be of use to liquefy nonliquefying semen, and that the resultant spermatozoa have a positive effect on pregnancy results.