Showing papers in "Journal of Andrology in 1998"

The molecular basis of sperm capacitation.

Abstract: Testicular sperm that have undergone spermatogeneis and spermiogenesis appear mature from a morphological standpoint but have acquired neither progressive motility nor the ability to fertilize a metaphase Il-arrested egg. In many species, progressive motility and fertilization competence are acquired during epididyma! transit, but complete fertilization capacity in vivo is only gained upon residence in the female reproductive tract for a finite period of time. The molecular and physiological events that confer on the sperm the ability to feftilize during residence in the female tract are collectively known as pacitation.” These maturational events can also be accomplished in vitro in defined media, the composition of which approximates the environment of the female reproductive tract. Although capacitation was discovered independently by Austin (1951, 1952) and Chang (1951, 1955) nearly one-half century ago, little is known to date about the molecular basis of this important event. This brief review will consider some of the recent findings made toward an understanding of capacitation using in vitro models. The purpose of this review is not to provide an exhaustive analysis of capacitation but is to offer an

Involvement of Reactive Oxygen Species in Human Sperm Arcosome Reaction Induced by A23187, Lysophosphatidylcholine, and Biological Fluid Ultrafiltrates

Abstract: Although recent evidence indicated that the production of reactive oxygen species (ROS) by human spermatozoa may be involved in the regulation of capacitation, very little is known about the role of ROS in the acrosome reaction. To address this issue, Percoll-washed spermatozoa were incubated in Ham's F-10 medium in the absence (no capacitation) or presence (capacitation) of fetal cord serum ultrafiltrate (FCSu) or progesterone. The effects of the ROS scavengers, superoxide dismutase (SOD), and catalase were then tested on the acrosome reaction induced by lysophosphatidylcholine (LPC), A23187, and ultrafiltrates from follicular fluid (FFu) and FCSu, as well as on the protein tyrosine phosphorylation associated with this process. 2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA)-amplified chemiluminescence was used to determine the extracellular superoxide (O 2 . ) production from spermatozoa. The observations that both SOD and catalase reduced (in the case of LPC) or totally prevented (in the other cases) the acrosome reaction of capacitated spermatozoa and that hydrogen peroxide (H 2 O 2 ) or ROS generated by the combination of xanthine and xanthine oxidase (O 2 . , which dismutates to H 2 O 2 ) triggered the acrosome reaction indicated the involvement of ROS in this process. In fact, capacitated spermatozoa in which the acrosome reaction was induced by LPC, A23187, and FFu produced more O 2 . than noncapacitated spermatozoa treated with the same agents. A23187 and LPC had minor effects on protein tyrosine phosphorylation of noncapacitated spermatozoa. However, these inducers caused a decrease in tyrosine phosphorylation of Triton-soluble proteins (mainly those of 37, 42, and 47 kDa) from capacitated spermatozoa, a decrease more pronounced in the presence of SOD. On the other hand, there was a marked increase in tyrosine phosphorylation of few proteins (70 to 105 kDa) from the Triton-insoluble fraction, which was partly reversed by SOD (in the case of LPC and A23187) or catalase (in the case of A23187), or abolished in the presence of the two antioxidants (in the case of A23187). These data indicate that the acrosome reaction is associated with an extracellular O 2 . generation by spermatozoa and that both O 2 . and H 2 O 2 may be involved in the regulation of this process. The mechanism by which these ROS act is unknown but may involve tyrosine phosphorylation of sperm proteins.

Human Sperm Capacitation Induced by Biological Fluids and Progesterone, but Not by NADH or NADPH, Is Associated With the Production of Superoxide Anion

Abstract: Recent evidence indicated that human sperm capacitation is associated with an increased production of superoxide anion (O2.−). To further study the role and importance of (O2.−). in capacitation, we investigated whether the (O2.−). generation is a general feature of capacitating spermatozoa, irrespective of the inducer used, and is correlated with capacitation levels and increased tyrosine phosphorylation of two sperm proteins (p105/p81). We also studied the time courses of (O2.−). production and action. Percoll-washed human spermatozoa were incubated in Ham's F-10 medium, supplemented or not supplemented with various capacitation inducers and in the presence or absence of superoxide dismutase (SOD). Sperm capacitation was measured by induction of the acrosome reaction with lysophosphatidylcholine, (O2.−). production was measured by chemiluminescence, and tyrosine phosphorylation was measured by immunodetection after electrophoresis and western blotting of sperm proteins. Progesterone and ultrafiltrates of human fetal cord serum, follicular fluid, and seminal plasma individually promoted sperm generation of (O2.−)., tyrosine phosphorylation of p105/p81, and capacitation. Fetal cord serum ultrafiltrate triggered a five-fold higher (O2.−). production than the other inducers (1,700 ± 300 and 300 to 400 mV/10s/8×10 cells, respectively), a phenomenon possibly associated with the higher potency of this fluid to promote sperm hyperactivation. The production of (O2.−). by spermatozoa was rapid and transient. SOD prevented sperm capacitation triggered by the inducers mentioned above, but only when SOD was added at the beginning of incubation, and not after 30 minutes, indicating that the (O2.−). initiates a chain of early events leading to sperm capacitation. NADH and NADPH (5 mM) triggered sperm capacitation and phosphorylation of p105/p81, but these processes were not prevented by SOD or catalase, nor were they associated with an increased (O2.−). production. Therefore, these cofactors appeared to act by mechanisms different from those used by the other inducers studied. The sperm enzyme responsible for the (O2.−). generation may be very different from the NADPH oxidase of neutrophils.

Rat testicular germ cells and epididymal sperm contain active P450 aromatase

Abstract: Although testosterone is the principal sex steroid produced by the testis, estrogen is known to be produced by both Leydig and Sertoli cells during different developmental periods. Additionally, evidence is unfolding to suggest that germ cells might also participate in the synthesis of estrogen within the male reproductive tract. We have recently reported that the messenger ribonucleic acid (mRNA) for P450 aromatase (P450arom), the enzyme that converts androgen to estrogen, is synthesized by rat germ cells. Therefore, the present study was conducted to determine which germ cell types synthesize active P450arom and to measure the activity of this enzyme in germ cells throughout spermatogenesis and in maturing sperm during epididymal transit. First, P450arom activity was measured in pachytene spermatocytes, round spermatids, and a mixture of round spermatids, elongating spermatids, and residual bodies using the tritiated water (3H2O) assay. Second, sperm isolated from different regions of the epididymis were assayed for P450arom activity. Sperm isolated from the caput epididymis with attached efferent ductules had the higher P450arom activity, whereas sperm isolated from the corpus and cauda epididymides had lower P450arom activity. The decrease in P450arom activity in cauda sperm was further confirmed by immunocytochemistry. On the basis of these observations, we conclude that rat testicular germ cells from pachytene spermatocytes through elongating spermatids and epididymal sperm contain active P450arom and that sperm lose aromatase activity as they mature during epididymal transit. Therefore, both post-pachytene rat germ cells and epididymal sperm are capable of estrogen synthesis and are an additional, potentially significant, source of estrogen in the male reproductive tract.

A pharmacokinetic study of injectable testosterone undecanoate in hypogonadal men

Abstract: Testosterone undecanoate (TU) provides testosterone (T) replacement for hypogonadal men when administered orally but requires multiple doses per day and produces widely variable serum T levels. We investigated the pharmacokinetics of a newly available TU preparation administered by intramuscular injection to hypogonadal men. Eight patients with Klinefelter's syndrome received either 500 mg or 1,000 mg of TU by intramuscular injection; 3 months later, the other dose was given to each man (except to one, who did not receive the 1,000-mg dose). Serum levels of reproductive hormones were measured at regular intervals before and after the injections. Mean serum T levels increased significantly at the end of the first week, from less than 10 nmol/L to 47.8+/-10.1 and 54.2+/-4.8 nmol/ L for the lower and higher doses, respectively. Thereafter, serum T levels decreased progressively and reached the lower-normal limit for adult men by day 50 to 60. Pharmacokinetic analysis showed a terminal elimination half-life of 18.3+/-2.3 and 23.7+/-2.7 days and showed a mean residence time of 21.7+/-1.1 and 23.0+/-0.8 days for the lower and higher doses, respectively. The area under the serum T concentration-time curve and the T-distribution value related to serum T concentration were significantly higher following the 1,000-mg dose than following the 500-mg dose. The 500-mg dose, when given as the second injection, yielded optimal pharmacokinetics (defined as mean peak T values not exceeding the normal range and persistence of normal levels for at least 7 weeks), suggesting that repeated injections of 500 mg at 6-8-week intervals may provide optimal T replacement. The mean serum levels of estradiol were normalized following the injections, and prolactin levels were normal throughout the study. Significant decrease of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels was observed, with the decrease in LH levels being more pronounced. There were no significant differences in serum LH and FSH levels between the two doses. Sex hormone-binding globulin (SHBG) levels before any T therapy were near the upper limit of normal for adult men and were reduced by approximately 50% just prior to the second dose of TU. The decreased SHBG levels produced by the first TU injection could have led to lower peak total T levels and to a more rapid clearance of T following the second TU injection. We conclude that single-dose injections of TU to hypogonadal men can maintain serum T concentration within the normal range for at least 7 weeks without immediately apparent side effects. It is likely that this form of T would require injections only at 6-8-week or longer intervals, not at the 2-week intervals necessary with currently used T esters (enanthate and cypionate). This injectable TU preparation may provide improved substitution therapy for male hypogonadism and, in addition, may be developed as an androgen component of male contraceptives.

Interaction Between Ca2+, Cyclic 3‘,5’ Adenosine Monophosphate, the Superoxide Anion, and Tyrosine Phosphorylation Pathways in the Regulation of Human Sperm Capacitation

Abstract: In order to fertilize the egg, spermatozoa must go through the capacitation process where they experience Ca 2+ uptake, increases in cyclic 3',5' adenosine monophosphate (cAMP) concentrations, superoxide anion production, and protein tyrosine phosphorylation. Although the importance of these processes has been described, the interactions between them, as well as the temporal sequence of these events, remain to be demonstrated. Previous studies from our laboratory have demonstrated that tyrosine phosphorylation of p105 and p81 (p105/81), the two major human sperm phosphotyrosine-containing proteins, was under cAMP and oxygen derivatives regulation. In the present study, we investigated the importance of intra- and extracellular Ca 2+ , as well as the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the phosphatase inhibitors calyculin A and okadaic acid, in the production of superoxide anion and p105/81 tyrosine phosphorylation. An increase in p105/81 phosphotyrosine content was observed when spermatozoa were incubated in the absence of extracellular Ca 2+ or with the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide. However, the human sperm capacitation inducer FCSu (ultrafiltrate of fetal cord serum) requires the presence of the extracellular Ca 2+ to induce capacitation, superoxide anion production, and tyrosine phosphorylation of p105/ 81, whereas free intracellular Ca 2+ had no effect on these last two processes. The production of superoxide anion by spermatozoa was stimulated by inhibitors of phosphodiesterases and serine/threonine phosphoprotein phosphatases. The tyrosine phosphatase inhibitor vanadate decreased by 40% the FCSu-stimulated superoxide anion production, although it had no effect when used alone. These results suggest that, during sperm capacitation, Ca 2+ induces an elevation in cAMP levels; this cAMP, through undefined serine/threonine protein phosphorylation, stimulates the generation of superoxide anion, which, in tum, causes the increase in p105/81 phosphotyrosine contents.

Triptolide: a potential male contraceptive.

Abstract: The antifertility effect of triptolide and other related compounds, isolated from Tripterygium wilfordii, has been demonstrated in male rats. The exact sites and mechanism of action of triptolide remain unknown. Our objectives were to determine whether triptolide at selected dose levels that induce infertility has any detrimental effects on the testes and to determine the sites and the possible mechanisms of its action. Groups of six adult male Sprague-Dawley rats were given oral administration of either vehicle (control group) or triptolide (50 or 100 microg/kg body weight) daily for 35 or 70 days. Body weight gain was normal in all treated groups. All six rats treated with a high dosage of triptolide were infertile during the second (63-70 days) mating trial. A lower dose (50 microg) of triptolide gave intermediate fertility values. Plasma levels of luteinizing hormone, follicle-stimulating hormone, testosterone, and intratesticular testosterone were not significantly different between control and triptolide-treated groups. Cauda epididymal sperm content was decreased by 68% and the motility, which averaged 58.2% in the control rat, was reduced to almost zero. No effects of triptolide were observed on testis and accessory organs weight, volumes of tubular lumen and the total Leydig cells, tubule diameter, and the number of Sertoli cells, spermatogonia, preleptotene (PL), and pachytene (P) spermatocytes. There were, however, modest but significant decreases in tubule volume and the number of round spermatids at stages VII-VIII. No changes in the germ cell apoptotic index measured at stages VII-VIII and XIV-I were noted between controls and rats rendered infertile with a high dose of triptolide. Thus, triptolide, at a dose level that induces complete infertility in the adult rats, has minimal adverse effects on the testes and acts primarily on the epididymal sperm making triptolide an attractive lead as a post-testicular male contraceptive.

Effects of Cryopreservation on Bull Sperm Head Morphometry

Abstract: Artificial insemination using cryopreserved semen is a common management tool of the contemporary livestock producer. However, cryopreservation is detrimental to sperm function and fertility, killing some 50% of the spermatozoa during the process. Prediction of cryopreservation damage from prefreeze samples remains elusive. Computer-automated sperm head morphometry was used in this study to determine the effects of cryopreservation on bovine sperm head morphometry. Semen was collected from 18 bulls and was divided. One portion was extended to 200 x 10(6) sperm/ml and a microscope slide was prepared, while the remaining portion was cryopreserved in a Triscitrate-yolk extender. After thawing, the cryopreserved samples were prepared on microscope slides. All slides were air dried and were stained with hematoxylin and rose bengal. The morphometric dimensions for length, width, width/length, area, and perimeter for a minimum of 200 sperm heads were analyzed from each slide by computer-aided sperm head morphometry analysis, and the mean measurements were recorded. Bull sperm heads were significantly (P < 0.01) smaller in cryopreserved spermatozoa than in the companion extended samples for length (8.56+/-0.07 vs. 8.63+/-0.08 microm), width (4.39+/-0.05 vs. 4.48+/-0.05 microm), area (28.42+/-0.07 vs. 29.14+/-0.08 microm), and perimeter (23.33+/-0.21 vs. 23.70+/-0.23 microm) for all bulls. Width/length was also different (0.513 vs. 0.519). In addition, differences (P < 0.05) were found within 14 of 18 bulls for at least four of the morphometric parameters. The percent change in measures after cryopreservation were correlated (P < or = 0.05) to the variability of the extended sample. Variations in sperm head measurements were lower (P < or = 0.05) in extended samples of the four bulls in which no changes occurred than in extended samples of the remaining 14 bulls. These data suggest that the variability in sperm head measurements of individual bulls, or ejaculates, may be an indicator of sperm cryosurvivability.

SP22: a novel fertility protein from a highly conserved gene family.

Abstract: Three nucleotide sequences encoding SP22, a protein originally identified in detergent extracts of cauda epididymal sperm, were isolated from a rat testis cDNA library. While two of these cDNA sequences differed only in their 5′ untranslated regions, a third cDNA was predicted to contain an additional 13 amino acids of coding sequence. Amino acid sequences obtained following Edman degradation of purified SP22 protein and cDNA sequence data both indicated that SP22 was a member of a highly conserved and widely expressed gene family found in organisms as diverse as human and Escherichia coli. Interestingly, while a 1-kb mRNA transcript was widely expressed in somatic tissues, a unique pattern of testicular expression was observed, including the appearance of a novel 1.5-kb transcript and an increase in the abundance of the 1-kb transcript during spermatogenic cell development. Anti-SP22 peptide antiserum was shown to recognize a family of 22-kDa proteins on western blots of detergent-extracted cauda epididymal sperm protein, suggesting that multiple charge variants of SP22 coexist. Moreover, affinity-purified anti-SP22 peptide immunoglobulin localized in a highly specific manner to the anterior-ventral surface of the equatorial segment of the sperm head. This is an extremely intriguing finding as SP22 was originally shown to be highly correlated with, and predictive of, the fertilizing ability of cauda epididymal sperm. Although no conclusive function has been attributed to any members of the SP22 gene family, the localization of SP22 over a discrete region of the sperm head suggests a pivotal role in sperm—egg interactions.

Environmental hormones and the male reproductive system.

Abstract: The growing number of reports documenting reproductive dysfunction in male humans and wildlife (Carlsen et al, have led to the hypothesis that hormonally active compounds in the environment may be adversely affecting the male reproductive system (Table 1). In this review, we define environmental hormones as compounds that mimic or antagonize the action of natural steroid hormones. Currently, four major classes of environmental hormones that interact directly with steroid hormone receptors are known: environmental estrogens, environmental antiestrogens, environmental antiproges-tins, and environmental antiandrogens. Environmental es-trogens include natural plant estrogens and synthetic chemicals. Synthetic chemicals such as pesticides, her-bicides, polychiorinated biphenyls (PCBs), plasticizers, and surfactant breakdown products are known to have estrogenic activity Recently, compounds with antiestrogemc, antiprogestigenic, or antian-drogenic activity have been identified, including dioxin (antiestrogenic; Safe et al, 1991); carbamate insecticides (antiprogestigenic; Klotz et al, 1997); and polyaromatic hydrocarbons, linuron, vinclozolin, and p,p'-DDE (antian-This review focuses on the kinds of reproductive dys-functions observed in humans and wildlife, with partic-Mimreview ular emphasis on the male reproductive system. The hypothesis that environmental chemicals can act as hormones , which disrupt normal sexual development during fetal life, is examined. Evidence from laboratory studies of whole animals and from in vitro studies that dissects the mechanism of interaction between environmental hormones and the vertebrate endocrine system is reviewed. Humans-The incidence of testicular cancer has increased significantly since the 1940s in European countries , ranging from a 50% increase A study of nine European countries concluded that the incidence increased 2-3% annually in the Nordic countries and 5% annually in Germany and Poland (Adami et al, 1994). All three studies found a much greater increase in the rate at which young men (<30 years old) developed testicular cancer. Currently, the only known risk factor for testicular cancer is cryptorchidism (Giwercman et al, 1993). Developmental abnormalities such as cryptorchi-dism and hypospadias have also been reported more frequently in recent years. However, criteria for diagnosing these conditions have changed over time, so this data must be interpreted with caution (Giwercman et a!, 1993). The most disputed change in human male reproductive health is the assertion that a significant, worldwide decline in semen quality (measured as sperm concentration) has occurred over the past 50 years (Carlsen et al, 1992). Retrospective studies in a Parisian sperm bank (Auger et al, 1995) and a London fertility clinic (Ginsburg et al, 1994) have supported this conclusion, although a study of three …

The effects of antifreeze peptide III (AFP) and insulin transferrin selenium (ITS) on cryopreservation of chimpanzee (Pan troglodytes) spermatozoa.

Abstract: We investigated the effects of antifreeze peptides (AFP) and insulin transferrin selenium (ITS) on the motility and membrane integrity of chimpanzee (Pan troglodytes) spermatozoa after chilling (0-5 degrees C) and thawing. The effects of three thawing procedures, in the presence or absence of AFP and ITS, on sperm motility and on the status of the plasma membrane and acrosome were also examined. During chilling, AFP and ITS seem mildly cytotoxic, as the progressive motility and velocity (curvilinear and straight line) declined significantly at AFP concentrations of 1, 10, and 100 microg/ml and at ITS concentrations of 1 and 10 microg/ml. However, at a concentration of 100 microg/ml, ITS was able to protect sperm during short-term hypothermic storage. Addition of AFP or ITS at 100 microg/ml to test egg yolk-glycerol extender during freezing significantly (P < 0.05) increased postthaw motility, plasma membrane integrity, and acrosome integrity. The mean (+/-SE) motility recovery rate increased from 28.9 +/- 3.9%, for the untreated control, to 59.2 +/- 5.8% and 67.8 +/- 7.4%, for ITS and AFP, respectively. The effects of the thawing procedure were influenced by the presence of AFP during the freezing cycle. An improved motility recovery rate of 67 +/- 4.2% was obtained when chimpanzee sperm frozen in test egg yolk-glycerol extender supplemented with AFP were thawed rapidly at 37 degrees C, compared to 47 +/- 5.2% and 44 +/- 8.2% for slow (23 degrees C) and ultrarapid (75 degrees C) thawing, respectively. The motility recovery after thawing of ITS-treated semen at 23 degrees C, 37 degrees C, or 75 degrees C was not significantly different. Semen frozen without AFP or ITS and thawed at 75 degrees C was seriously (P < 0.05) damaged. This study provides evidence that AFP- or ITS-supplemented semen extender improves postthaw sperm motility in the chimpanzee.

Cytoplasmic Extrusion and the Switch From Creatine Kinase B to M Isoform are Completed by the Commencement of Epididymal Transport in Human and Stallion Spermatozoa

Abstract: Although in several species there is a relationship between epididymal sperm transport and fertility, in human in vitro fertilization (IVF), spermatozoa recovered from the caput epididymidis or even the rete testis are fertile. We studied two objective markers of sperm maturity in the sperm of men and stallions: creatine kinase (CK) concentrations, which are a measure of cytoplasmic retention in immature spermatozoa, and the ratio of CK-M and CK-B isoforms (% CK-M/[CK-M + CK-B]), which is proportional to the incidence of mature sperm. The CK markers and the fertilizing function are closely related: Immature sperm with cytoplasmic retention do not bind to the zona, because during cytoplasmic extrusion, the sperm plasma membrane is also remodeled. We examined whether changes in sperm CK values are still ongoing during epididymal transport, or if cellular maturation is completed prior to the arrival of sperm in the caput epididymidis. The incidences of mature sperm in human caput and corpus epididymidis (studied in six men with obstructive azoospermia of various pathogeneses) were (mean ± SEM) 55.7 ± 2.2 and 49.3 ± 7.6%, respectively; and the sperm CK-M ratios in the caput epididymidis of three men were 72, 75, and 70%, values that are similar to those of ejaculated sperm. In four segments of the proximal and distal epididymis of three stallions (the origin of sperm was also verified by the position of the cytoplasmic droplet) and in ejaculate of five stallions, the incidences of mature sperm were 88.2 ± 6.2, 89.0 ± 6.7, 90.3 ± 7.8, 87.6 ± 5.9, and 86.7 ± 0.8%, and the respective CK-M ratios were 75.0 ± 8.7, 84.2 ± 2.9,87.9 ± 1.2, 92.5 ± 1.5, and 69.3 ± 3.5%. There were no differences in the incidences of mature and immature spermatozoa or in CK-M ratios among sperm arising from the various epididymal regions or from the ejaculate in men or stallions. Thus, the cellular maturation events in sperm, as detected by the CK markers, are completed by the time the sperm commences epididymal transport. These findings are in agreement with the IVF fertility of sperm aspirated from the male reproductive tract. The data may also suggest that the primary role of sperm epididymal transport in men is to remodel the plasma membrane to enhance sperm functional integrity in the diverse environments of the male and female reproductive tracts prior to fertilization.

Follicle-stimulating hormone is required for the initial phase of spermatogenic restoration in adult rats following gonadotropin suppression.

Abstract: The role of follicle-stimulating hormone (FSH) in adult rat spermatogenesis is unclear. Although exogenous testosterone (T) restores spermatogenesis following gonadotropin-releasing hor- mone (GnRH) immunization or T plus estradiol (TE) treatments, an assessment of the independent action of T and FSH was not pos- sible, as exogenous T treatment maintains serum FSH levels. We have used passive immunization against FSH to determine whether T alone is capable of reinitiating spermatogenesis after chronic and acute FSH withdrawal. Adult rats received T-filled Silastic implants 6 cm (T6) or 8 cm (T24) in length for 7 days in combination with eithera polyclonalsheep antisera raised against rat FSH (FSHAb, 2 mg/kg SC daily)or controlsheep immunoglobulin (ConAb) after either GnRH immunization (12 weeks) or TE treatment (9 weeks). The neutralizing capacity of the FSHAb was determined using a FSH in vitro bioassay; this analysis demonstrated that administration of FSHAb in vivo reduced FSH levels by >90%. Testes were fixed and germ ceO number per testis quantified using the optical dissector. GnRH immunization reduced spermatogonia, pachytene spermato- cytes, and round spermatids to 50, 13, and <1% of normal, respec- tively. T6 and T24 Silastic implants with the inclusion of the FSHAb did not increase the number of spermatogonia, pachytene spermat- ocytes, and round spermatids (50, 15, and 1% of normal, respec- tively). T6+ConAb treatment increased spermatogonial, pachytene spermatocyte, and round spermatid numbers to 74, 30, and 3% of normal, respectively (P < 0.05). No further increases were seen with T24 implants. TE treatment suppressed pachytene spermatocytes and round spermatids to 33 and 1% of normal, respectively (P < 0.05). T6+FSHAb treatment did not increase the number of pachy- tene spermatocytes and round spermatids (36 and 8%, respective- ly), whereas T6+ConAb treatment increased pachytene spermato- cyte and round spermatid number to 50 and 28% of normal, re- spectively (P < 0.05). T24+ FSHAb treatment increased the number of pachytene spermatocyte and round spermatids (56 and 22% of normal, respectively; P < 0.05), whereas T24+ConAb treatment in- creased these cells forms to 79 and 31% of normal, respectively. In conclusion, T alone is unable to restore spermatogenic cell popu- lations in the setting of chronic FSH withdrawal. Although acute FSH withdrawal markedly impairs the restoration process, higher doses of T can partially compensate for the lack of FSH. These data sug- gest that FSH is important for the initial phase of spermatogenic restoration.

Antioxidant potential of human serum albumin: role in the recovery of high quality human spermatozoa for assisted reproductive technology.

Abstract: Human serum albumin (HSA) is being considered as an alternate media for sperm enrichment in assisted reproductive technology (ART) because of recent concern with the use of Percoll. In this study, we compared HSA and Percoll for 1) sperm recovery, 2) reactive oxygen species scavenging potential, and 3) effects on total oxidative stress to spermatozoa. The spermatozoa-enriched fractions obtained from Percoll (80%:40%) and HSA (12%) were monitored for sperm motility, viability, hypoosmotic swelling test (HOST), and adenosine triphosphate (ATP) levels. The effect of superoxide anions (O 2 .) on donor human spermatozoa was observed in the presence of either HSA or Percoll media. A combination of luminol and the Cypridina luciferin analog 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo(1,2-α)pyrazin-3-one hydrochloride was used as a highly sensitive chemiluminescence probe in our hypoxanthine and xanthine oxidase-based assay for O 2 . . Sperm membrane total oxidative stress was determined by measuring levels of the prostanoid 8-iso-Prostaglandin F 2α (8-Iso-PGF 2α ). Significant differences in sperm parameters between the Percoll-enriched spermatozoa (motility 60% ± 4%, viability 56% ± 6%, and HOST 73% ± 7%) and those enriched with HSA (motility 84% ± 5%, viability 85% ± 4%, and HOST 84% ± 3%; P < 0.01) were observed. Adenosine triphosphate levels were significantly higher, by almost 50%, in samples processed with HSA than with Percoll (P = 0.03). The dismutation rate of O 2 . in HSA (slope -6.8) was significantly lower than in Percoll (slope -87.0; P < 0.01). Sperm motility and ATP levels decreased at a slower rate after treatment with O 2 . in the presence of HSA when compared to Percoll; moreover, spermatozoa in HSA regained partial motility after 2 hours, whereas spermatozoa in Percoll were immobilized. No significant differences in 8-iso-PGF 2α levels in spermatozoa enriched by eιther HSA or Percoll were observed. We conclude that the HSA sperm enrichment procedure improves the recovery of higher quality spermatozoa compared to Percoll and, because of its antioxidant properties, may be useful in processing high leukospermia semen samples for ART purposes.

Influence of centrifugation regimes on motility, yield, and cell associations of mouse spermatozoa.

Abstract: Mouse sperm are exceptionally sensitive to mechanical forces associated with pipetting and mixing. This characteristic raised the question of the sensitivity of mouse sperm to centrifugation, a step necessary in the removal of cryoprotectants and a common component in the general manipulation of sperm suspensions for experimental purpose. Epididymal spermatozoa from ICR mice were isolated and manipulated to minimize pipetting and mixing damage. The centrifugal accelerations studied were 200, 400, 600, and 800 x g (measured with a stroboscope) for 5, 10, or 15 minutes of centrifugation time. The number of cells and the number of motile cells were counted. The percent motility and longevity, total yield, and motile yield were calculated. Centrifugation at 200 and 400 x g for short times (5 minutes) caused only a small loss in either immediate or 2.5-hour motility, but centrifugation at 600 and 800 x g for 15 minutes produced up to a fivefold loss. Low speed/short time centrifugation pelleted only about half of the cells; the others were lost when the supernatant was removed. The maximum number of motile sperm (motile yield) was obtained at intermediate centrifugal forces (approximately 400 x g for 10-12 minutes), and it is the total number of motile sperm (and not the percent motility) that is important in the use of cryopreserved sperm to regenerate cryopreserved mutant lines. Relative centrifugal force and centrifugation time exhibit reciprocity (e.g., 200 x g for 10 minutes produces similar results to 400 x g for 5 minutes). The spermatozoa must be centrifuged under carefully defined conditions to minimize the damage and to maximize the recovery of viable cells.

Evidence for Nitric Oxide Regulation of Hamster Sperm Hyperactivation

Abstract: Involvement of reactive oxygen species has been im- plicated in the process of hyperactivation and capacitation of sperm. Nitric oxide has recently been found to function both as an intracel- lular and extracellular messenger, with its synthetic enzyme found in several cell types, including male and female genital tract organs. The objective of the present study was to investigate the role of nitric oxide in hamster sperm hyperactivation. Caudal epididyrnal contents of mature golden hamster sperm were diluted with human tubal me- dium supplemented with a sperm motility preparation. Inhibitors of nitric oxide synthase (nitro-L-arginine, methyl-L-arginine, and 1,3- phenylene-bis(1 ,2-ethenediyl)-bis-isothiourea) were added to incu- bation media in various doses. Alternatively, a nitric oxide donor, sodium nitroprusside, was used. The percentage motile and grade of movement were recorded at intervals encompassing the normal period of capacitation and hyperactivation. Acrosomal status was evaluated by phase contrast microscopy. Inhibition of nitric oxide synthesis did not affect motility during early capacitation but dra- matically inhibited later hyperactivation. An inactive stereo-enanto- mere of the inhibiting drug had no effect. Addition of nitric oxide to nonstimulated sperm induced hyperactivation in a similar time course. In conclusion, nitric oxide plays a significant role in hyper- activation of hamster epididymal sperm.

Androgen Regulation of the Pem Homeodomain Gene in Mice and Rat Sertoli and Epididymal Cells

Abstract: Although the role of homeodomain transcription factors during embryogenesis is well known, their developmental function in postnatal animals is only beginning to be understood We examined the regulation and expression pattern of Pem, a homeodomain protein that may regulate androgen-dependent events in the testis and epididymis Immunohistochemical analysis showed that Pem protein is expressed selectively in the nuclei of Sertoli cells during the androgen-dependent stage of the seminiferous epithelium cycle in vivo RNase protection analysis revealed that a proximal promoter was responsible for androgen-dependent mouse Pem expression in testis and epididymis in vivo, whereas a distal promoter was used in placenta The mouse Pem gene was expressed at approximately 10-fold higher levels in the testis than in the epididymis; conversely, the rat Pem gene was expressed at >10-fold higher levels in the epididymis than in the testis Because androgen-binding protein has been proposed to transport androgens from the testis to the epididymis, we tested whether the > or = 20-fold higher levels of androgen-binding protein expression in the rat, compared to that of mouse, are responsible for the differential expression of Pem in these two rodent species Studies with androgen-binding protein transgenic mice demonstrated that the species-specific difference in androgen-binding protein expression is unlikely to be responsible for the species-specific difference in Pem expression We found that androgen is necessary but not sufficient for Pem expression, since purified Sertoli cells rapidly down-regulated Pem transcripts in culture, regardless of the presence of testosterone We conclude that Pem gene expression in Sertoli cells requires other cell types or cellular factors in addition to androgen

Semen Donor Selection by In Vitro Sperm Mobility Increases Fertility and Semen Storage in the Turkey Hen

Abstract: Commercial turkey production relies on the artificial insemination (AI) of pooled semen. However, semen quality ultimately depends on the quality of individual ejaculates. The purpose of this study was to evaluate semen from individual toms by means of an objective sperm-mobility assay. Semen was then pooled by mobility phenotype and inseminated into hens, and the percentages of fertile and hatched eggs were determined after egg incubation. To indirectly evaluate hens' sperm storage, we determined the number of sperm holes in the perivitelline layer (PL) of freshly laid eggs. Semen from individual ejaculates (two trials, total of 169 toms) was evaluated by use of the sperm-mobility test (SMT). Semen was diluted to 1 x 10(9) sperm/ml, in prewarmed N-tris-[hydroxymethyl] methyl-2-amino-ethanesulfonic acid (TES)-buffered saline, and was placed over 3 ml of a 2% (w/v) Accudenz solution at 41degrees C. After a 5-minute incubation period, the cuvette was placed in a densimeter, and percentage transmission was recorded after 1 minute. Semen samples from toms ranked, according to sperm mobility, in the highest 10% and the lowest 10%, after three evaluations, were pooled by group and were used to inseminate hens weekly (trial 1: n = 20 hens/group, for 10 weeks, AI dose 150 x 10(6) spermatozoa inseminated fresh and after 24-hour in vitro storage at 5 degrees C; trial 2: n = 60 hens/group, for 16 weeks, AI dose = 75 x 10(6) spermatozoa inseminated fresh). Each week, eggs from day 6 post-AI were evaluated for holes in the PL, vestiges of acrosomal induced hydrolysis. Spermatozoa from toms of different mobility phenotypes were also evaluated individually, for sperm chromatin structure and motility variables, by use of the Hobson Sperm Tracker. Toms characterized by high and low in vitro sperm-mobility phenotype were categorized as "high mobility" and "low mobility," respectively. The percentage of fertile eggs from hens inseminated with semen from the high-mobility toms was higher than the percentage of fertile eggs from the low-mobility group, in each trial (95.8+/-1.3% vs. 90.4+/-2.2%, and 88.7+/-4.0% vs. 82.4+/-0.4%, trials 1 and 2, respectively; P < 0.05). More sperm holes were observed in the PL of eggs fertilized by the high-mobility toms than in the PL of eggs fertilized by the low-mobility toms (P < 0.05). No differences in susceptibility of sperm nuclear DNA to denature in situ, as measured by the flow-cytometric sperm chromatin-structure assay, were detected between toms of differing mobility phenotypes. Sperm-motility variables, straight-line velocity, and average-path velocity were significantly greater for high-mobility toms compared to low-mobility toms (P < 0.05). Sperm-mobility differences between toms (detected by means of the SMT) influenced sperm storage, as indicated by the number of sperm in the PL and by the percentage of fertile eggs produced.

Detection of Human Glandular Kallikrein, hK2, as Its Precursor Form and in Complex With Protease Inhibitors in Prostate Carcinoma Serum

Abstract: Forms of human glandular kallikrein (kK2) in pros- tate carcinoma serum were identified using monoclonal antibod- ies specific for hK2 and prohK2. Recombinant mammalian hK2, prohK2, and prostate = specific antigen (PSA) were utilized to confirm the specificity of monoclonal antibodies for hK2 and the lack of reactivity with PSA. In prostate cancer patient sera con- taining high levels of hK2 (>100 ng/ml), hK2 exists as a complex with a1-antichymotrypsin with a molecular weight of 90 kDa. The kallikrein also exists as a 32-kDa free form, which includes the precursor pro form of hK2. The relative amount of complex and free hK2 varied, but in most sera examined the 32-kDa form pre- dominated. Recombinant hK2 readily formed complexes with a2- macroglobulin when the two proteins were incubated together as well as when hK2 was spiked into female serum.

Ethnic Differences in Testicular Structure and Spermatogenic Potential May Predispose Testes of Asian Men to a Heightened Sensitivity to Steroidal Contraceptives

Abstract: Spermatogenesis in Asian men appears to be more susceptible to suppression by steroidal contraceptives administered in clinical trials than spermatogenesis in Caucasian men. The objective of this study was to determine whether ethnic differences exist in testicular structure and spermatogenic potential that might predispose Asians to a high sensitivity to steroidal contraceptives. Testes from 12 Chinese men were compared to those from 8 Hispanic men and 12 non-Hispanic Caucasian men of ages 29+/-3, 30+/-2, and 29+/-3 years, respectively. Testes were fixed by vascular perfusion with glutaraldehyde, further fixed in osmium, embedded in Epon, and evaluated by stereology using 0.5-microm sections stained with toluidine blue. Homogenates of fixed testes were evaluated for the number of Sertoli cells and the daily sperm production based on pachytene primary spermatocytes (PDSP) or spermatids with spherical nuclei (DSP). Paired parenchymal weight was less (P < 0.05) in Chinese men than in Hispanic or Caucasian men. The PDSP per gram of parenchyma was lower (P < 0.05) and the DSP per gram tended to be lower in Chinese men than in other groups. The histologic appearance, volume density, and length per man of seminiferous tubules were the same among the ethnic groups; however, the diameter of seminiferous tubules was less (P < 0.05) in Chinese than in Hispanic or Caucasian men. The PDSP per man and the DSP per man were lower (P < 0.05) in Chinese than in Hispanic or Caucasian men. The number of Sertoli cells per gram was higher (P < 0.05) in Chinese or Caucasian men than in Hispanic men, but the number of Sertoli cells per man was lower (P < 0.05) in Chinese men than in Hispanic or Caucasian men. Sertoli cell function, measured as the number of germ cells accommodated by a single Sertoli cell, was lower (P < 0.05) in Chinese men than in Caucasian men. The volume density of Leydig cell cytoplasm was greatest (P < 0.05) in Chinese men, but the number of Leydig cells was similar among the ethnic groups. Hence, smaller testes coupled with reduced Sertoli cell number and function and reduced daily sperm production could predispose Asian men to have a heightened negative response of testes to steroidal contraceptives, as compared to Caucasian men. Dampening (by exogenous androgens) of any physiological benefit to spermatogenesis that a high volume density of Leydig cell cytoplasm may bestow on the human testis (that Asian men may have evolved to require) would exacerbate ethnic differences in the spermatogenic response to hormonal contraceptives.

Vitamin E Deficiency Causes Incomplete Spermatogenesis and Affects the Structural Differentiation of Epithelial Cells of the Epididymis in the Rat

Abstract: The effects of vitamin E deficiency on the rat testis and epididymis were examined in a light- and electron-microscopic analysis Various groups of animals were made vitamin E-deficient, beginning at postnatal day 10, via their lactating mothers, until day 21, when they were separated from their mothers The groups were maintained thereafter on either a vitamin E-deficient or a normal diet (controls) The vitamin E-deficient animals of group A, sacrificed at day 42, revealed testes that were normal in appearance, with a full complement of germ cells when compared to their controls (group B) Group C, however, sacrificed at day 48, revealed major abnormalities in the testes, unlike both their controls (group D) and normal, untreated animals (group E) Spermatogenesis was incomplete; the most advanced cell type was predominantly step-7 spermatids However, many of these cells, as well as earlier spermatids, appeared to undergo degeneration, evidenced by large pale areas in their nuclei, disrupted acrosomes, and a cytoplasm with uncharacteristic organelles Multinucleated cells, characterized by their chromatoid bodies as spermatids, were often seen in the seminiferous tubule lumen Sertoli cells were normal in appearance, except for numerous, large lipid droplets in their basal region, at stages I-VIII; in appropriate controls (group D), such droplets were absent at these stages These lipid inclusions presumably represented the final breakdown products of the late spermatids, which were phagocytosed by Sertoli cells between days 42 and 48 However, numerous germ cells, often recognized as round spermatids, and multinucleated cells were noted in the epididymal lumen, which indicates that such cells were spared from Sertoli cell phagocytosis These data suggest that vitamin E plays a key role in the maintenance and survival of spermatids In the epididymis, vitamin E deficiency resulted in principal, narrow, and apical cells that showed a poorly developed secretory and endocytic apparatus at days 42 (group A) and 48 (group C), unlike those of normal, untreated animals (group E) On the other hand, clear cells of groups A and C showed a highly developed endocytic apparatus in the cauda region only, whereas in the caput and corpus regions, endocytic apparatuses were small and undifferentiated, unlike those of group E Thus, in the epididymis, vitamin E plays a role in the structural differentiation of principal cells along the entire epididymis, whereas, in the case of clear cells, its role is region-specific Readministration of vitamin E to the diet restored a normal appearance to both the testis and the epididymis, which indicates that the effects on these tissues are reversible Taken together, these data indicate that vitamin E plays important roles in maintaining the viability of the spermatid population and in allowing epithelial epididymal cells to acquire their fully differentiated structural appearance

Apoptosis Pattern Elicited by Several Apoptogenic Agents on the Seminiferous Epithelium of the Adult Rat Testis

Abstract: Spontaneous germ cell death during spermatogenesis is an important event, and the usefulness of the seminiferous epithelium as an in vivo model to study apoptosis has been evidenced. Nevertheless, the response of the testis to apoptogenic agents has not been analyzed. This study was designed to determine germ cell sensitivity to induction of apoptosis and to provide baseline data on the testis response to several apoptogenic agents. Induced apoptosis was assessed by in situ DNA 3'-end labeling and quantified at every stage of the spermatogenic cycle. The shortest response time for every agent was established based on morphological and quantitative criteria. Our results show significantly increased incidence of germ cell deaths after all treatments, mainly at stages I, XII, and XIV. These specific stages coincide with those at which the greatest numbers of spontaneous germ cell deaths occur in control animals. Moreover, the rapid and highly specific response of germ cells to all the apoptogenic agents used in the present study indicate that apoptosis must be tightly regulated at these stages of the seminiferous epithelium. As a consequence, we propose that the disruption of apoptosis control might be an important determinant for idiopathic male infertility.

Hormonal regulation of spermatogenesis in the hypophysectomized rat: quantitation of germ-cell population and effect of elimination of residual testosterone after long-term hypophysectomy

Abstract: Spermatogenesis continues after long-term hypophysectomy (Hx), but massive cell degeneration prevents seminiferous tubules from attaining the full complement of cells. One objective of this study was to determine the vulnerable sites for completion of spermatogenesis in long-term Hx rats. It is now known that Leydig cells continue to secrete small amounts of androgen after Hx. A second objective was to determine the cellular sites that are maintained by residual androgen secreted by Leydig cells post-Hx. Two groups of adult animals were utilized. Both groups were Hx for 36 days, but one group of rats received the androgen antagonist flutamide during the 26th through the 36th day of Hx (10 days). Germ-cell numbers were quantified using a method that allowed their expression as numbers of cells present per hour of development. In the long-term Hx rat, the germ-cell population increased to preleptotene, but the divisions that led to preleptotene were inefficient due to cell degeneration. Subsequent to preleptotene, there was a gradual loss in cells such that there were few germ cells remaining by steps 9-13. Flutamide given to Hx rats did not result in a significant difference in the numbers of intermediate and type B spermatogonia or significant differences in progenitor cells. A significant and major depression of cell numbers in Hx-flutamide-treated rats occurred in the cell division of type B spermatogonia to form preleptotene spermatocytes. There was a less dramatic, although significant, depression of cell numbers in Hx-flutamide-treated animals that occurred from preleptotene until late pachytene as well as an increased loss of round spermatids at midcycle (step 5-6). These data demonstrate that cell loss after long-term Hx occurs at numerous phases of spermatogenesis. The data also demonstrate that the presence of residual androgen action after long-term Hx results in enhanced germ-cell survival. Although the major blockage in cell viability occurs at midcycle steps in the long-term Hx rat, there are several other hormone-sensitive phases of spermatogenesis.

Hormonal regulation of spermatogenesis in the hypophysectomized rat: cell viability after hormonal replacement in adults after intermediate periods of hypophysectomy

Abstract: A quantitative analysis of germ-cell populations in normal, hypophysectomized (Hx), and Hx-hormone-treated animals was undertaken during periods of regression that were characterized as intermediate, between short-term and long-term regression of the testis. Twenty-one groups of adult rats were administered either follicle-stimulating hormone (FSH), growth hormone, thyroid-stimulating hormone (TSH), or testosterone (T) in various doses and combinations. The dosage of T administered was less than that expected to achieve maximum testis weight. Flutamide and Casodex were used to compete with androgen binding to receptors in Hx animals, as it is known that small amounts of androgen are secreted in the absence of pituitary stimulation. Follicle-stimulating hormone, T, and TSH all significantly maintained testis weight as compared with Hx controls, although FSH and T, singly or in combination, were the most effective. Contamination of the TSH preparation with trace amounts of FSH was apparently responsible for the slight maintenance of testis weight. A novel assay for determination of the numbers of viable germ cells was used in a subset of these groups to determine the cellular sites of FSH and T action. Numbers of type A spermatogonia were lowered after Hx and were maintained by either FSH or T or a combination of these hormones. Other phases of germ-cell development most susceptible to FSH and/or T were the successive conversions of type A spermatogonia to intermediate spermatogonia, intermediate spermatogonia to type B spermatogonia, preleptotene spermatocytes to pachytene spermatocytes, and early pachytene spermatocytes to intermediate maturity pachytene spermatocytes during early and midcycle phases of pachytene spermatocyte development. Germ-cell loss during meiosis and virtually every phase of spermatid development was largely prevented by FSH or T or a combination of these hormones. Thus, in testes in advanced stages of regression, both FSH and T were capable of preventing cell loss, suggesting that both hormones can affect the survival of the same cell type. The present study demonstrated that FSH can partially compensate for lowered T levels. The combined administration of FSH and T was more effective in preventing overall cell degeneration than either hormone alone. Unlike the initial phase of spermatogenesis, in which there is a largely midcycle loss of germ cells due to Hx, the loss of cells during testis regression is more widespread and impacts several cell types in more than one stage of the spermatogenic cycle.

Extender components and surfactants affect boar sperm function and membrane behavior during cryopreservation.

Abstract: To determine how the individual components of extenders affected boar sperm function and membrane structure and to test a new surfactant's cryoprotective ability, boar sperm were cryopreserved in straws in BF5 extender plus or minus egg yolk plus or minus glycerol plus or minus a surfactant (Orvus ES Paste [OEP] or various concentrations of Pluronic F-127) After thawing, sperm function and fluidity of the isolated head plasma membrane (HPM) were determined Total motility and adenosine triphosphate content (a measure of viability) were superior postthaw in sperm extended in egg yolk plus glycerol (P < 005); neither surfactant improved function Egg yolk plus any other ingredients improved normal acrosome morphology, whereas a combined measure of motility and normal acrosome morphology was better in the presence of 033% OEP or 01% Pluronic F-127 (P < 005 vs controls) Head plasma membrane was isolated from freshly collected spermatozoa and spermatozoa cryopreserved in the various extenders Membrane fluidity was monitored with the probes cis-parinaric acid (cPNA), transparinaric acid (tPNA), and 1,6-diphenyl-1 ,3,5-hexatriene (DPH) The cPNA and the DPH monitor the fluidity of gel and liquid-crystalline areas of the membrane, whereas the tPNA preferentially monitors the gel-phase domains of the membrane Additionally, DPH monitors the hydrophobic core of the bilayer In the HPM from fresh sperm, the fluidity of each domain changed over time in a manner unique to that domain, and the behavior of the DPH domain varied among boars The fluidity dynamics of each domain responded uniquely to cryopreservation The cPNA domain was unaffected, the tPNA domain was altered by four of the eight extenders, and all extenders affected the fluidity of the DPH domain Membrane structure was significantly correlated with cell function for sperm cryopreserved in extenders that preserved viability and motility Sperm cryopreserved in egg yolk plus glycerol plus either OEP or 01% Pluronic F-127 functioned best when the bulk domains were less fluid initially and the gel domain solidified more slowly Therefore, the behavior of domains in the HPM of boar spermatozoa is affected by cryopreservation and is related to the postthaw function of boar sperm cryopreserved in different extenders

Regulation of sperm function by protein tyrosine phosphorylation in diverse wild felid species.

Abstract: Protein tyrosine phosphorylation is associated with sperm capacitation and the acrosome reaction in several mammalian species. Changes in phosphorylation of a 95-kDa protein in human, mouse, and domestic cat spermatozoa are known to be influenced by capacitation and exposure to zona pellucida (ZP) proteins. We previously reported diminished phosphorylation of 95- and 160-kDa proteins in spermatozoa from teratospermic cats, compared with normospermic domestic cats. To determine if these proteins and mechanisms are present in other species in the phenotypically diverse Felidae family, we examined the relationship between tyrosine-phosphorylated sperm proteins and sperm morphology in the leopard cat (approximately 65% normal sperm/ejaculate), tiger (approximately 65%), clouded leopard (approximately 15%), and cheetah (approximately 30%). Furthermore, we investigated the involvement of cyclic adenosine monophosphate (cAMP) in the regulation of sperm protein tyrosine phosphorylation. Specifically, we assessed the following: 1) presence of tyrosine-phosphorylated proteins in sperm extracts; 2) changes in protein tyrosine phosphorylation after sperm capacitation and swim-up separation; 3) impact of tyrosine kinase inhibition on leopard cat sperm protein phosphorylation and ZP penetration; and 4) involvement of a cAMP-dependent pathway in the regulation of protein tyrosine phosphorylation. Immunoblotting analysis with anti-phosphotyrosine antibody (PY20) indicated that a 95-kDa protein was present in all four species. Additional phosphorylated proteins were detected in the leopard cat (145- and 175-kDa proteins), tiger (185-kDa protein), clouded leopard (160- and 190-kDa proteins), and cheetah (115- and 155-kDa proteins). Sperm capacitation in vitro increased phosphorylation of one or more proteins in the leopard cat, tiger and clouded leopard, but not in the cheetah. Although swim-up separation increased the proportion of morphologically normal spermatozoa in the clouded leopard and cheetah, no changes were observed in phosphorylation of the 95-kDa sperm protein. Thus, phosphorylation of the 95-kDa protein appeared to be related to the condition of teratospermia. Exposing leopard cat spermatozoa to the tyrosine kinase inhibitor, tyrphostin, reduced (P < 0.05) phosphorylation of the 95- and 145-kDa proteins, as well as ZP penetration, without affecting sperm motility. Similarly, when spermatozoa were incubated in the presence of cAMP analogs or active and inactive stereoisomers of cAMP, phosphorylation of sperm proteins was either stimulated or inhibited. Together, these data suggest that protein tyrosine kinase mechanisms appear conserved within the family Felidae and are regulated by a cAMP/protein kinase A pathway.

Evidence for cross-talk between Sertoli and germ cells using selected cathepsins as markers.

Abstract: To examine whether proteases are possibly involved in cellular migration and/or spermiation when developing germ cells translocate across the seminiferous epithelium during spermatogenesis, in situ hybridization was used to localize messenger RNA (mRNA) transcripts of cathepsin L, D, and S in the epithelium at different stages of the spermatogenic cycle in the rat. Cathepsin L mRNA was found to localize almost exclusively near the basal lamina of the epithelium. At stages VI and VII of the cycle before spermiation, the signal of cathepsin L mRNA was so intense that it formed a complete dark precipitate near the basal lamina encircling the entire tubule. At stage VIII, the expression of cathepsin L was completely abolished, and no staining of cathepsin L mRNA was seen in the epithelium. The mRNA of cathepsin D and S was found near the basal lamina, a finding consistent with their localization in Sertoli cells and possibly primary spermatocytes in almost all stages, but peaked at stages VII-IX and VII-VIII of the cycle, respectively, at the time before and during spermiation. These results illustrate the possible involvement of these proteases in facilitating germ cell movement and spermiation. To examine whether germ cells express any of these cathepsin genes, spermatocytes with a purity of greater than 95% were isolated from 15-day-old rat testes by Percoll gradient centrifugation for reverse transcriptase-polymerase chain reaction. It was found that primary spermatocytes expressed multiple cathepsin genes, including cathepsin B, C, D, H, L, and S. Furthermore, the expression of cathepsin L by germ cells isolated from 15-day-old rats (largely spermatocytes and spermatogonia) can be stimulated by Sertoli cell-enriched culture medium in a dose-dependent manner, but not by germ cell-conditioned medium. These results reveal that germ cell function can be regulated by Sertoli cells. Moreover, these results suggest that germ cells may play an active role in the overall testicular protease expression. Also, we present evidence suggesting there is cross-talk between Sertoli and germ cells, since the expression of cathepsin L in each cell type is regulated by one another via either soluble factors or cell-cell contact.

Androgen binding protein secretion and endocytosis by principal cells in the adult rat epididymis and during postnatal development.

Abstract: Androgen binding protein (ABP) has been shown to be secreted by Sertoli cells and to be actively taken up by the efferent ducts and proximal caput epididymidis and, yet, to be present at high concentrations in epididymal fluids. In the present study, ABP was immunolocalized by light microscopy in epithelial cells of the efferent ducts and epididymis of adult rats and during postnatal development and by electron microscopy in specific organelles within these cells. In adults, the efferent ducts actively endocytosed Sertoli cell-derived ABP. In the epididymis, principal cells displayed a variable staining reminiscent of a checkerboardlike pattern, with cells being intensely, moderately, or weakly reactive throughout their cytoplasm or unreactive. In the electron microscope, reactive cells displayed a labeling of their Golgi apparatus and secretory vesicles indicative of an epididymal-secreted form of ABP. However, labeling was also noted over endosomes of principal cells, but only of the initial segment and intermediate zone, which, along with labeling of coated pits and vesicles, indicated that ABP was also endocytosed by principal cells of these regions. The postnatal study revealed that principal cells attained an adultlike staining pattern indicative of secretion in a region-specific manner at different ages, suggesting that ABP secretion is regulated by different factors. Ligation of the efferent ducts of 15-day-old animals revealed no reaction along the entire epididymis in animals sacrificed at later ages, suggesting the importance of luminal testicular factors in its regulation during development. In addition, as in the adult, ABP was also endocytosed by principal cells, but only in the initial segment and intermediate zone. Taken together, the present results indicate that secretion of ABP occurs along the entire epididymis, whereas endocytosis is region specific. The functional role of ABP in the epididymis in relation to sperm maturation is discussed.

Quantification of apoptotic testicular germ cells in normal and methoxyacetic acid-treated mice as determined by flow cytometry.

Abstract: Several studies have reported the occurrence and significance of programmed cell death (apoptosis) of testicular germ cells in mammals. In those studies, apoptotic germ cells were identified by morphological criteria or by in situ end labeling (TUNEL) and were enumerated from histological sections by semi-quantitative and time-consuming techniques. In the present study, we have established a flow cytometric technique for quantification of TUNEL-positive cells in the mouse testis. Groups of five adult mice each received 0, 650, or 1300 mg/kg (IP) of methoxyacetic acid (MAA), and testes were collected 24 hours later. MAA is known to induce germ cell apoptosis in rodent testes. MAA induced a significant (P < 0.01) dose-dependent decline in the percentage of pachytene spermatocytes (4C cells). DNA strand breaks generated by the activation of endogenous endonuclease in the apoptotic germ cells were detected by the in situ labeling of the 3'-OH termini with biotinylated dUTP in the presence of terminal deoxynucleotidyl transferase (TUNEL technique). Histologically, TUNEL-positive germ cells were observed in control testes, and the number of these cells was visibly increased following MAA exposure. As determined by flow cytometry, four cell populations contained TUNEL-positive cells: 1C cells (round spermatids), 2C cells (mainly spermatogonia), S-ph cells (spermatogonial cells and preleptotene spermatocytes synthesizing DNA [the S-phase]), and 4C cells (primary spermatocytes). Analysis of the percentages of TUNEL-positive cells within each population yielded values of 1.57+/-0.23% for 1C cells, 1.65+/-0.27% for 2C cells, 6.26+/-1.03% for S-ph cells, and 3.24+/-0.39% for 4C cells. Hence, a substantial proportion of proliferating cells are undergoing apoptosis during normal spermatogenesis. The overall incidence of apoptotic cells among all testicular cells was around 2%. At 650 mg per kilogram of body weight, MAA induced a fourfold to eightfold increase (P < 0.001) in the percentage of TUNEL-positive cells, compared with saline-treated controls, and, overall, 17% of testicular cells were apoptotic. This effect of MAA was most pronounced for S-ph and 4C cells, with 25-30% of cells being affected in each of those populations. At 1300 mg per kilogram of body weight, MAA had no further effect. These quantitative data demonstrate that 1) in the normal testis, it is mainly proliferating cells that undergo apoptosis, and 2) MAA induces primary spermatocyte loss by germ cell apoptosis.