Differentiated structure and function of primary cultures of monkey oviductal epithelium.

We have established well-differentiated, polarized cultures of monkey oviductal epithelium. Oviductal epithelial cells were isolated by protease digestion and plated on collagen-coated, porous cell culture inserts. About 5 d after plating, cells developed detectable transepithelial electrical resistance of up to 2000 Omega.cm(2) (an index of tight junction formation) and transepithelial voltages of up to 20 mV (an index of vectorial transepithelial ion transport). Measurements of short-circuit current in Ussing chambers indicated that active secretion of Cl was the major transepithelial active ion transport process, and that this was stimulated by elevation of either cAMP or Ca(i). Furthermore, estimates of the volume of mucosal liquid were consistent with Cl secretion mediating fluid secretion. Various microscopical methods showed that the cultures were densely ciliated and contained mature secretory cells. Transport across the oviductal epithelium determines the composition of the oviductal fluid, and the study of the relevant transport processes will be greatly enhanced by well-differentiated cultures of oviductal epithelium of the kind established here.

The carbohydrate structure of DEFB126, the major component of the cynomolgus Macaque sperm plasma membrane glycocalyx.

Based on the amino-acid sequence of the macaque epididymal secretory protein, ESP 13.2 (Q9BEE3/AJ236909), it has now been classified as beta-defensin DEFB126. DEFB126 is one of the five beta-defensins with genes that are clustered along chromosome 20pl3, and all five proteins have an extended carboxy terminus that continues beyond the 6-cysteine beta-defensin core region. This 60-amino acid carboxyl tail extension of the DEFB126 molecule is extraordinarily rich in threonine and serine (40%), many of which appear to be likely candidates for having O-glycosylation. DEFB126 has been shown to coat the entire surface of cynomolgus macaque sperm as they move through the corpus/caudal region of the epididymis. It is a major glycocalyx barrier to the external environment and is retained until the completion of capacitation. Sperm exposed to fluorescein-conjugated poly-L-lysine or Alexa488-histone showed a very uniform fluorescent labeling pattern over the entire sperm surface, almost identical to that observed with anti-DEFB126 Ig label. Sperm surface components that were released following treatment with caffeine/cAMP (in vitro capacitation) were blotted and probed with three different lectins which are known to recognize terminal sialic acid residues, and all three recognized the 35 kDa DEFB126 band. Neuraminidase treatment of sperm shifted the molecular weight of DEFB126 from 34-36 kDa to approximately 38-40 kDa and removed or greatly inhibited sialic acid-specific lectin recognition. O-Glycanase treatment alone was ineffective at removal of the oligosaccharides, but prior treatment with neuraminidase was sufficient to enable the O-glycanase treatment to effectively change the apparent molecular weight to 10 kDa, confirming that a major portion of the molecular mass is associated with the carbohydrate portion. Western blots of neuraminidase-treated DEFB126 showed strong recognition with a number of lectins that identify beta-galactose and also lectins that recognize the N-acetylgalactosamine-serine/threonine, the proposed connection site of O-glycosylation. All of the lectins that recognized DEFB126 on Western blots were used to fluorescently probe sperm. The fluorescent patterns that were observed with poly-L-lysine, Alexa488-histone, sialic acid-specific lectins, and galactose-specific lectins showed even distributions over the entire sperm surface and the patterns were identical to sperm labeled with anti-DEFB126 Ig, and all but the antibody did not recognize neuraminidase-treated sperm.

Identification of a novel GPI-anchored CRISP glycoprotein, MAK248, located on the posterior head and equatorial segment of cynomolgus macaque sperm.

To identify a sperm-surface component that is highly antigenic, we immunized female cynomolgus macaques with glycosylphosphatidylinositol (GPI)-anchored sperm surface proteins that were released following treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Five different adjuvants were used in combination with the PI-PLC-released proteins, and three of these proteins (24, 48, and 53 kDa) were shown to be potent antigens for immunization of female monkeys. The 53 kDa protein was found to be a surface coating protein and not a GPI-anchored protein. Polyclonal antibodies to the 24 kDa protein and the 48 kDa protein were produced in rabbits. The two antibodies recognized both proteins on Western blots. The same rabbit antibodies recognized 28, 18, and 10 kDa bands on a Western blot of chemically reduced PI-PLC-released proteins, suggesting that the 48 kDa protein is a dimer of the 24 kDa protein, which we refer to as MAK248. Rabbit polyclonal antibodies developed to reduced fragments of the 24 kDa protein showed that the 18 and 10 kDa bands are proteolytic peptide fragments of the 24 kDa protein. Screening of tissues from male macaques showed that MAK248 is expressed only in the epididymis. Microsequencing of two proteolytic fragments of the 18 kDa component showed 100% amino acid homology to a 233 deduced amino acid sequence previously identified in human testes genome. Antibodies to MAK248 recognized a 24 kDa protein released from human sperm exposed to PI-PLC. Antibodies to MAK248 recognized the equatorial segment and posterior head regions of capacitated cynomolgus macaque sperm. Structural analysis suggests that MAK248 is a novel CRISP protein and a member of the CAP (CRISP, Ag 5, PR-1) family of proteins. Based on amino acid sequence homology, it is possible that MAK248 functions as a protease inhibitor.

Immunization of female cynomolgus macaques with a synthetic epitope of sperm-specific lactate dehydrogenase results in high antibody titers but does not reduce fertility.

Previous studies have reported reduced fertility in female baboons immunized with a synthetic peptide derived from the sperm-specific isozyme of lactate dehydrogenase (LDH-C). In this study, a similar approach was used to immunize female cynomolgus macaques with the same peptide sequence (bC5-19) conjugated to a T-cell epitope from tetanus toxin (TT). Twelve female monkeys were immunized with bC5-19:TT delivered with Ribi MPL adjuvant vehicle, and 10 control female monkeys were injected with the adjuvant vehicle only. All 12 females in the treatment group developed LDH-C-specific serum antibodies as measured by ELISA, but anti-LDH-C antibodies were not detected in vaginal fluids of the immunized animals. After 4 months of timed mating immediately following the immunizations, five of the ten immunized females became pregnant, as did six of the ten control females. Anti-sera from both pregnant and nonpregnant bC5-19:TT-immunized females recognize a single band at 35 kDa on Western blots of whole sperm extracts, and purified Igs from the same sera localize along the principle piece of the flagellum of permeabilized sperm.

Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides.

Because of its location on the sperm surface and its multiple functions during fertilization, the PH-20 protein is a potential target for contraceptive vaccines. Cynomolgus macaques were immunized using four different adjuvants together with synthesized peptides or recombinant proteins representing selected regions of macaque PH-20. The synthesized peptide (amino acids 387-412, designated Peptide 4) was used as a linear molecule in a 1:1 ratio with a peptide sequence of tetanus toxoid, as well as a multiple antigenic peptide (MAP) matrix held together by scaffolding lysine residues. In the MAP construct, the ratio of Peptide 4 to tetanus peptide was 4:1. To circumvent the poor production of recombinant PH-20 in bacterial cells, two truncated forms of the molecule were expressed in Escherichia coli, G18 (encoding amino acids 143-510) and E10 (encoding amino acids 291-510). The adjuvants were Montanide ISA 51, Titermax Gold, Syntex adjuvant formulation (SAF), and QS-21. All of the antigen/adjuvant combinations produced significant immune responses as measured by ELISA. The circulating antibodies from immunized animals recognized macaque sperm surface PH-20 on Western blots and were shown by indirect immunofluorescence to bind to the surface of macaque sperm. Montanide and Titermax were associated with higher titers of anti-PH-20 antibodies than QS-21 and SAF adjuvants. Immunization with Titermax, however, resulted in sterile abscesses in 4 of 8 animals injected. We conclude that antigens derived from synthesized peptides and recombinant proteins representing selected regions of the PH-20 molecule can be used as vaccine components in combination with the adjuvant Montanide to elicit a significant sperm-directed antibody response in immunized macaques.

Soybean trypsin inhibitor as a probe for the acrosome reaction in motile cynomolgus macaque sperm.

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to acrosome react with calcium ionophore A23187 in the presence of SBTI and were fixed for ultrastructural observation. Transmission electron microscopy (TEM) revealed secondary labelling of anti-SBTI-IgG with colloidal gold in association with the acrosomal matrix and fused membranes of sperm undergoing the acrosome reaction, but gold labelling was not observed on acrosome-intact sperm. When SBTI was conjugated with the fluorochrome Alexa 488, labelled (acrosome-reacted) sperm showed bright fluorescence that ranged from a patchy or punctate appearance to solid labelling over the region of the acrosomal cap. Following treatment with ionophore, the percentages of total acrosome-reacted sperm (motile and non-motile) as assessed with Alexa-SBTI, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and TEM were 54.6%, 51.6% and 61.5%, respectively. Measures of acrosomal status with FITC-PSA and Alexa-SBTI were highly correlated (r = 0.94; n = 3). Macaque zonae pellucidae were co-incubated with activated sperm for 1 min and then rinsed in medium containing Alexa-SBTI and immediately observed with epifluorescence microscopy. The mean percentage of Alexa-SBTI-labelled (acrosome-reacted) motile sperm bound to the zona was 45.7 +/- 14 (range: 22-80.4%; n = 4). Fewer than 1% of the motile sperm in suspension surrounding the zonae were acrosome-reacted. Alexa-SBTI had no effect on sperm motility, survival, or zona binding capability.

Effect of protein kinase C stimulators on zona pellucida binding and the acrosome reaction of macaque sperm.

Macaque sperm require treatment with dibutyryl cAMP (dbcAMP) and caffeine (termed activators; ACT) to fully capacitate in vitro. Previous studies have shown that treatment with ACT also increases sperm binding to the macaque zona pellucida and enhances the induction of acrosome reactions of the bound sperm. This study investigated whether phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-sn-glycerol (DOG), which stimulate protein kinase C (PKC), can also increase sperm binding to the zona pellucida and/or enhance induction of the acrosome reaction of bound sperm. Cynomolgus macaque sperm were centrifuged through 60% Percoll and then were washed with modified Biggers, Whitten and Whittingham (BWW) medium and incubated for 2.5 h at 37 degrees C with 5% CO2, ACT, PMA, or DOG was added to sperm during the last 15-30 min of incubation. Sperm were evaluated immediately after incubation for motility and acrosomal status. Macaque oocytes were coincubated with stimulated sperm suspensions in BWW medium for 30 sec (pulse). Half of the oocytes were removed to sperm-free medium and further incubated for 1 h (chase). When assessed after the pulse period, sperm-zona binding increased significantly after ACT treatment compared to that in untreated controls. DOG but not PMA treatment had a similar effect on sperm binding PMA, DOG, and ACT treatment increased the percentage of acrosome reactions in sperm bound to the zona following the 30-sec pulse compared to the percentage in controls. Inactive analogs of PMA and DOG had no effect on sperm-zona binding or the percentage of acrosome reactions (% AR) of bound sperm.(ABSTRACT TRUNCATED AT 250 WORDS)

Separate effects of caffeine and dbcAMP on macaque sperm motility and interaction with the zona pellucida.

Capacitation of macaque sperm with caffeine and dbcAMP is required for fertilization in vitro. This study determined the separate effects of caffeine and dbcAMP on sperm-zona pellucida binding and the acrosome reaction of zona bound sperm. Semen from 6 cynomolgus macaques was washed through 60% Percoll, resuspended, and washed with BWW media and incubated for 2.5 hr. Caffeine, dbcAMP (2 mM each), or both (1 mM each) were added to aliquots of the sperm suspensions. Immature macaque oocytes were placed into drops of sperm suspensions, coincubated with sperm for 30 sec, and either fixed immediately or removed to sperm-free media and incubated 1 hr before fixation. There were no significant differences between groups in the percentage of live, acrosome-reacted sperm in suspension. Treatment with caffeine and dbcAMP or with caffeine alone, significantly increased the number of sperm bound to each zona pellucida (96 +/- 16 and 81 +/- 17, respectively) compared to control and dbcAMP treatment (15 +/- 4 and 28 +/- 13). However, treatment with dbcAMP, alone and with caffeine, resulted in a higher percentage of acrosome-reacted sperm on the zona (15.2 +/- 2.1 and 9.0 +/- 0.6) than control or caffeine treatment (3.0 +/- 1.4 and 2.4 +/- 0.5). Effects on sperm motility consistent with hyperactivation were detected only when both caffeine and dbcAMP were present. Although both caffeine and dbcAMP are presumed to increase or to produce the same effect as increased intracellular cAMP levels, these compounds have different effects on the ability of sperm to bind to the zona and to undergo the acrosome reaction.

Comparison of a fluoresceinated lectin stain with triple staining for evaluating acrosome reactions of dog sperm.

In this study a technique which utilized a fluoresceinated lectin and a fluorescent supravital stain was compared with the conventional triple stain technique for evaluating the viability and acrosomal status of dog sperm. Ten semen samples obtained from 6 normal beagle dogs were evaluated after incubation in vitro with canine capacitation medium. Sperm viability and acrosomal status were assessed at 0, 4, and 7 hours of incubation. Both staining techniques were capable of detecting the increase in spontaneous acrosome reactions which occurs during in vitro capacitation of dog sperm. High positive correlations were observed between the fluorescent stain and the triple stain in the mean values for the percentage of viable sperm and for the percentage of acrosome-reacted sperm among the viable sperm (r = 0.91, r = 0.97, respectively). However, the fluorescent staining techniques could be carried out much more rapidly than the triple stain technique, and the slides prepared with fluorescent stain were more easily scored because of the higher intensity and greater consistency of staining. These characteristics make the fluoresceinated lectin and the fluorescent supravital stain superior for evaluating acrosome reactions and viability of dog sperm.

Sperm-zona pellucida interaction in cynomolgus and rhesus macaques.

These experiments were carried out to establish and validate an in vitro system for studying macaque sperm-zona pellucida interaction. Sperm of rhesus and cynomolgus macaques were capacitated in vitro and incubated with cryopreserved zonae pellucidae. Homologous gamete incubations were tested, as well as cross-species combinations. Approximately 25% of macaque sperm bound to the zonae acrosome reacted within 1 minute of gamete coincubation, although the percentage of acrosome reactions in the sperm suspension was less than 1%. There was a small but consistent increase in the percent of acrosome reactions of zona sperm after an additional hour of incubation in sperm-free media. Similar results were obtained in the cross-species experiments, suggesting that zonae from the two macaque species can be used interchangeably in sperm-zona binding assays. Differences in the physiologic characteristics of the sperm of the macaque species were demonstrated. Cynomolgus sperm required activation with caffeine and dibutyryl cyclic adenosine monophosphate (dbcAMP) in order to bind to the zonae. Rhesus sperm were able to bind to the zonae and acrosome react in the absence of activators, although both sperm binding and percentage of acrosome reactions increased with the addition of activators. Large numbers of sperm from both macaque species bound to the zonae of hamster oocytes after treatment with activators, but the bound sperm did not acrosome react. These experiments demonstrate the importance of evaluating the acrosomal status of sperm when sperm-zona binding assays are performed with macaque gametes.

Cryopreservation of spermatozoa from cynomolgus monkeys (Macaca fascicularis).

Three egg-yolk diluents, which have been used successfully in cryopreservation of human spermatozoa, were compared for their ability to protect macaque semen against cryodamage. TEST (Tes + Tris + egg yolk), TEST with 20% skim milk (TSM), and egg yolk-citrate (EYC), each with 3 or 5% glycerol were compared using 12 ejaculates from 6 male cynomolgus macaques. Computer-aided analysis of sperm motion was used to determine the percentage motility (%M), curvilinear velocity (VCL), and linearity (LIN) of spermatozoa after thawing. The supravital stain Hoechst 33258 and a fluoresceinated pea lectin were used to determine the % of viable spermatozoa with intact acrosomes. TSM and TEST were superior to EYC in terms of % M and of % viable, acrosome-intact spermatozoa. TSM and TEST produced equivalent VCL and LIN values, while EYC had clearly reduced VCL and LIN. There were no interactions between diluent and glycerol level. The 3% glycerol level gave superior results to 5% glycerol for %M. EYC, which is widely used for cryopreservation of human spermatozoa, was not suitable for cynomolgus monkey semen. Artificial insemination with semen cryopreserved in TSM resulted in a healthy, full-term infant.

Ultrasound-guided transfundal uterine sperm recovery from Macaca fascicularis.

Previous studies from this center have indicated that the cynomolgus macaque (Macaca fascicularis) may serve as a model for human sperm interaction with the cervix and uterus. In some macaque species, transcervical aspiration of the uterine contents carries a significant risk of disturbing the cervical milieu due to the serpentine nature of the cervix. The only alternatives have been surgical procedures such as laparotomy or laparoscopy. In this paper, we report our experience with a new technique for ultrasound-guided sampling of spermatozoa in the macaque uterus. Twenty adult female cynomolgus macaques were monitored for menses (first day of menses = day 1), and one mating per cycle was allowed on day 10, 11, or 12. In one group of ten animals, cervical mucus was sampled at 3 or 18 hr postcoitus (pc) and ultrasound-guided uterine aspiration was performed at 24 hours pc. In a second group of ten monkeys, uterine aspiration was at six hr pc and sperm numbers and motility were counted in the uterine fluid. Uterine fluid was obtained from fourteen of twenty monkeys. Pregnancy occurred in ten of the twenty experimental cycles. Ultrasound-guided uterine aspiration appears to be a reliable method for the evaluation of sperm transport in female macaques. The correlations between uterine sperm recovery and cervical mucus sperm populations are discussed. The high conception rate in treatment cycles indicates that this procedure can be performed without apparent risk to pregnancy.