Ovarian and immunological responses to alternating exogenous gonadotropin regimens in the ocelot (Leopardus pardalis) and tigrina (Leopardus tigrinus)

Exogenous gonadotropins frequently are used to stimulate ovarian follicular growth and ovulation in mammalian species, including felids. However, repeated exogenous gonadotropin treatment can result in decreased ovarian responsiveness due to antibody formation. In this study, our objectives were to assess the effectiveness of alternating gonadotropin regimens on ovarian responses in ocelots and tigrinas, and investigate the humoral immune responses to these gonadotropins in each species. Females were treated four to six times with alternating equine chorionic gonadotropin (eCG)/human chorionic gonadotropin (hCG) and porcine follicle stimulating hormone (pFSH)/luteinizing hormone (pLH) regimens at 4‐month intervals. With each treatment, the females were evaluated laparoscopically to assess ovarian follicular development and recover oocytes from mature follicles. Blood was collected before each treatment and at laparoscopy. Overall, the ocelots averaged more (P<0.05) follicles and corpus luteum (CL) (6.8±0.8; mean±SEM) per stimulation than the tigrinas (2.3±0.4), but the percentage of mature oocytes (mean range=54–55%) did not differ (P<0.05). Within species, both gonadotropin regimens were equally effective (P>0.05) in inducing follicular growth and oocyte maturation. The total number of ovarian structures and oocyte maturation percentages did not decrease (P<0.05) in either species with sequential stimulations. Although the percentage of blood samples containing anti‐gonadotropin immunoglobulins increased (P<0.05) with sequential treatment, the presence of positive titers did not cause a decrease (P<0.05) in ovarian responsiveness. In summary, the female ocelots and tigrinas continued to respond to these alternating ovarian stimulation protocols after repeated use, despite the formation of anti‐gonadotropin antibodies in some of the females. These findings suggest that the use of alternating gonadotropin regimens may permit more intensive reproductive management of these endangered cat species for conservation. Zoo Biol 00:1–14, 2005. © 2005 Wiley‐Liss, Inc.     

Ovarian response to repeated administration of alternating exogenous gonadotropin regimens in the ocelot (Leopardus pardalis) and tigrinus (Leopardus tigrinus)

Exogenous gonadotropins are used to stimulate ovarian follicular growth and ovulation in mammalian species, including wild cats. However, successes in application of assisted reproduction techniques in nondomestic felids have been sparse. Our objectives were to assess the effectiveness of alternating gonadotropin regimens on ovarian responses. Five adult female ocelots and four adult female tigrinus were treated four to six times, using alternating eCG/hCG and pFSH/pLH at 4-month intervals. Laparoscopies were done to assess follicular development and to collect oocytes from matures follicles. The average number of follicles and corpus luteum (CL) per stimulation was higher in ocelots (7.0 +/- 0.8; mean +/- S.E.M.) than in tigrinus (2.5 +/- 0.4; P < 0.05), but the percentage of mature oocytes did not differ between the two species (mean range, 54-55%). Within species, both gonadotropin regimens were equally effective in inducing follicular growth and oocyte maturation. The total number of ovarian structures and oocyte maturation percentages did not decrease in either species with sequential stimulations. In summary, female ocelots and tigrinus continued to respond to repeated alternating ovarian stimulation protocols. In conclusion, the use of alternating gonadotropin regimens may permit more intensive reproductive management in these endangered cats.     

Seasonal analysis of semen characteristics,serum testosterone and fecal androgens in the ocelot (Leopardus pardalis), margay (L. wiedii) and tigrina (L. tigrinus)

Captive adult male ocelots (Leopardus pardalis, n = 3), margays (L. wiedii, n = 3) and tigrinas (L. tigrinus, n = 4) in two locations in southern Brazil were studied for 14 consecutive months to evaluate the effect of season on testicular function. Reproductive evaluations, including testicular measurements, electroejaculation and blood collection were conducted monthly. Fecal samples were collected weekly for androgen metabolite analysis to assess testicular steroidogenic activity. Ocelots had the highest number of motile spermatozoa in the ejaculate (114.7+/-15.8 x 10(6); P < 0.05), the highest percentage of morphologically normal spermatozoa (82.4+/-1.2%; P < 0.05) and the highest concentration of fecal androgens (1.71 vs. 0.14 microg/g; P < 0.05). Margays and tigrinas had lower numbers of motile spermatozoa (23.4+/-2.8 x 10(6), 74.2+/-8.9 x 10(6), respectively), lower percentages of morphologically normal spermatozoa (57.4+/-2.8, 59.2+/-3.5%, respectively), and lower fecal androgen concentrations (0.15+/-0.01, 0.23+/-0.01 microg/g, respectively). Serum testosterone concentrations were similar among the three species. Fecal androgen concentrations were not affected by season, with the exception of the ocelot where concentrations were higher (P < 0.05) in the summer. Ejaculates were collected throughout the year; however, peaks in average sperm production were observed during the summer for all species. In summary, this study has identified several species differences in male testicular traits among ocelots, margays and tigrinas. Results of longitudinal reproductive assessments suggest males of each species are capable of breeding throughout the year.     

Prey refuges as predator hotspots: ocelot (Leopardus pardalis) attraction to agouti (Dasyprocta punctata) dens

We tested the hypothesis that prey refuges attract predators, leading to elevated predator activity in the vicinity of refuges. We used camera traps to determine whether the spatial activity of a predator, the ocelot (Leopardus pardalis), was biased toward refuge locations of its principal prey, the agouti (Dasyprocta punctata). We radio-tracked agoutis at night to locate active refuges and compared the activity of ocelots between these refuges and surrounding control grid locations. We found that ocelots visited the area near agouti refuges significantly more often and for longer periods of time than control locations, and that they actively investigated the refuge entrances. Both occupied and unoccupied refuges were visited, but the duration of inspection was longer at occupied refuges. As the ocelots could probably not see the agoutis within the refuges, olfaction likely cued foraging ocelots. Two refuges were repeatedly visited by the same ocelots on different days, suggesting spatial memory. Overall, our results suggest that predators can be attracted to prey refuges or refuging prey. The benefits to prey of staying nearby a refuge would thus be counterbalanced by higher likelihoods of predator encounter. This should stimulate prey to use multiple refuges alternatingly and to not enter or exit refuges at times of high predator activity.     

Comparison of the ability of progesterone and heat solubilized porcine zona pellucida to initiate the porcine sperm acrosome reaction in vitro

It has been previously shown that progesterone can initiate the acrosome reaction (AR) of capacitated human and hamster sperm in vivo. We report here that progesterone can initiate a morphologically normal AR in porcine sperm that have undergone capacitation in a Hepes-buffered medium in vitro. In addition, we have compared the abilities of progesterone and heat-solubilized porcine zona pellucida (zona) to initiate the porcine sperm AR. Capacitated porcine sperm were treated with 1 m?g/ml progesterone, 150 m?g/ml porcine zona, or solvent control for 10 min. After treatment, sperm were incubated with the supravital dye Hoechst 33258, fixed and the acrosomal status determined in the previously viable sperm by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutini (FITC-PSA). There was no significant difference between the percentage of AR initiated by zona compared to that initiated by progesterone. In order to determine whether there was a synergistic interaction between the two AR initiators, both were added simultaneously to capacitated porcine sperm at optimal (1 m?g/ml progesterone, 150 m?g/ml zona) and suboptimal (75 ng/ml progesterone and 75 m?g/ml zona) concentrations. Simultaneous addition of the two AR-initiators at the two concentrations stimulated an additive AR-initating response, rather than a synergistic one. Several possible explanations for the additive results are discussed. © 1994 Wiley-Liss, Inc.     

Evaluation of sperm functional attributes in relation to in vitro sperm-zona pellucida binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality

The objective of this study was to evaluate sperm functional attributes in relation to in vitro sperm-zona binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality. Frozen-thawed forty-eight ejaculates from eight Surti buffalo bulls (six ejaculates/bull) obtained by artificial vagina were used. Frozen semen from each bull was thawed, pooled, and subjected for sperm functional (six replicates) and in vitro fertilization (four replicates) tests. The progressive forward motility, plasmalemma functional integrity assessed by fluorogenic [6-carboxyfluorescein diacetate (CFDA), and propidium iodide (PI)], hypoosmotic swelling (HOS), and hypoosmotic swelling-Giemsa (HOS-G) test, mitochondrial membrane potential, sperm nuclear morphology, the number of sperm bound to zona and cleavage rate differed significantly (P < 0.05) between bulls. When the animals were grouped based on cleavage rate (group I, >40% cleavage rate, n = 5, and group II, <40% cleavage rate, n = 3), in vitro fertility parameters and all the sperm functional attributes except sperm nuclear morphology differed significantly (P < 0.05). The proportions of sperm with functional plasmalemma in the tail and intact acrosome assessed by HOS-G test (25.33, range: 17.48–40.27) were significantly (P < 0.001) lower than the functional plasmalemma in the tail assessed by HOS test (39.80, range: 27.85–54.67). The number of sperm bound to zona had significant correlations with the mitochondrial membrane potential (r = 0.90, P < 0.01) and plasmalemma integrity (fluorogenic, r = 0.74 and HOS, r = 0.79, P < 0.05) and HOS-G, r = 0.87, P < 0.01). The cleavage rate had significant (P < 0.05) correlations with the mitochondrial membrane potential (r = 0.70) and plasmalemma integrity measured by HOS-G test (r = 0.68). The present study indicates that these attributes could represent important determinants of buffalo sperm quality influencing cleavage rate.     

Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes

Background

Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes.

Methods

Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20°C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO₂ in air at 38°C and 100% humidity in the presence of 5 × 10⁶ fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM).

Results

Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger.

Conclusion

In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20°C are less suitable for use in feline ZBA.     

Porcine zona pellucida ZP3α glycoprotein mediates binding of the biotin-labeled Mr 55,000 family (ZP3) to boar sperm membrane vesicles

The two M_r 55,000 glycoproteins, ZP3α and ZP3b?, of porcine zona pellucida copurify as a preparation designated ZP3. Gamete binding assays have implicated ZP3α, but not ZP3b?, as participating in sperm-zona recognition events. We now report that boar sperm contain membrane-associated binding sites with specificity for ZP3α. Biotin-labeled (b-) preparations of ZP3 bind to intact boar sperm in a saturable manner, with localization on the anterior head region. Membrane vesicles obtained from capacitated sperm by nitrogen cavitation retain b-ZP3 binding sites as determined by an enzyme-linked method employing alkaline phosphatase-conjugated strepavidin. In competitive binding assays using b-ZP3 (0.1μg/ml) as probe, heat-solubilized zonae and ZP3 were effective competitors, whereas the nonzona molecules fetuin and fucoidin were not. Digestion of ZP3 with endo-b?-galactosidase, an enzyme that trims polylactosamines, enhanced its affinity for membrane receptors. In contrast treatments such as chemical deglycosylation, pronase digestion, or disruption of disulfide bonds abolished the ligand activity of ZP3. Finally, purified ZP3α was an at least 100-fold better antagonist than purified ZP3b?. The results demonstrate that binding of b-ZP3 to isolated boar sperm membranes is mediated by sperm receptors with specificity for the ZP3α macromolecular component and reveal a complex contribution of both carbohydrate and protein moieties toward the ligand activity of this sperm adhesive zona molecule. © 1993 Wiley-Liss, Inc.     

Bovine sperm raft membrane associated Glioma Pathogenesis-Related 1-like protein 1 (GliPr1L1) is modified during the epididymal transit and is potentially involved in sperm binding to the zona pellucida

Glioma pathogenesis‐related 1‐like protein1 (GliPr1L1) was identified by liquid chromatography‐tandem mass spectrometry analyses of proteins associated to bovine sperm lipid raft membrane domains. This protein belongs to the CAP superfamily including cysteine‐rich secretory proteins, Antigen 5 and pathogenesis‐related 1 protein. PCR analysis revealed that GliPr1L1 is expressed in testis and, at a much lower level, all along the epididymis. Western blotting showed a similar distribution of GliPr1L1 in testicular and epididymal tissue extracts. In the epididymal lumen, GliPr1L1 was associated with the maturing spermatozoa and epididymosomes all along the excurrent duct but was undetectable in the soluble fraction of epididymal fluid. The protein was detectable as multiple isoforms with a higher MW form in the testis and proximal caput. Treatments with PNGase F revealed that N‐glycosylation was responsible of multiple bands detected on Western blots. These results suggest that the N‐glycosylation moiety of GliPr1L1 is processed during the transit in the caput. Western blots demonstrated that GliPr1L1 was associated with the sperm plasma membrane preparation. GliPr1L1 is glycosyl phosphatidyl inositol (GPI) anchored to caput and cauda spermatozoa as demonstrated by the ability of phosphatidylinositol specific phospholipase C to release GliPr1L1 from intact sperm cells. Lipid raft membrane domains were separated from caput and cauda epididymal spermatozoa. GliPr1L1 was immunodetectable in the low buoyant density fractions where lipid rafts are distributed. GliPr1L1 was localized on sperm equatorial segment and neck. In vitro fertilization performed in presence of anti‐GliPr1L1 showed that this protein is involved in sperm–zona pellucida interaction. J. Cell. Physiol. 227: 3876–3886, 2012. © 2012 Wiley Periodicals, Inc.     

Sperm binding and penetration of the zona pellucida in vitro but not sperm-egg fusion in an Australian marsupial, the brushtail possum (Trichosurus vulpecula)

Sperm capacitation and in vitro fertilisation (IVF) have been achieved in most eutherian mammals and American marsupials under relatively simple culture conditions. In contrast sperm capacitation in Australian marsupials has not been achieved in vitro and attempts at IVF have previously been characterised by a complete lack of sperm-zona pellucida (ZP) binding. Recently, co-culture of sperm with oviduct epithelial cell monolayers or with oviductal explant conditioned media has been shown to prolong the viability and motility of brushtail possum spermatozoa, as well as to induce capacitation-associated changes such as transformation of sperm to the T-shape orientation. In this study we report that these in vitro produced T-shaped sperm, and in vivo derived T-shaped sperm flushed from the oviduct of artificially inseminated possums as a control, are able to bind to and penetrate the ZP of approximately 25% of eggs recovered from PMSG/LH-superovulated possums in vitro. Development of ZP receptivity and penetrability towards sperm was also identified as a major factor affecting the outcome of IVF. Neither in vivo nor in vitro derived T-shaped sperm were able to bind to or penetrate the ZP if eggs were obtained from animals that were treated with pLH less than 76 h after PMSG. Thus this study provides preliminary evidence for the necessity of sperm-oviduct epithelial cell interactions for capacitation in Australian species and lends further support to the suggestion that the T-shape head orientation is indicative of sperm capacitation. Despite the occurrence of sperm-ZP binding and penetration, sperm-egg membrane fusion and egg activation were not observed. Although the factor(s) responsible for the lack of sperm-egg membrane fusion in the possum have not been identified it is possible that egg capacity for membrane fusion develops independently of zona receptivity and is defective in these eggs, or alternatively that membrane fusion requires strictly defined ionic conditions which are not provided by the IVF media used in this study.     

PDC-109 (BSP-A1/A2) promotes bull sperm binding to oviductal epithelium in vitro and may be involved in forming the oviductal sperm reservoir

Sperm reservoirs have been found in the oviducts of several species of mammals. In cattle, the reservoir is formed by the binding of sperm to fucose-containing glycoconjugates on the surface of oviductal epithelial cells. A fucose-binding molecule was purified from sperm extracts and identified as PDC-109 (BSP-A1/A2), a protein that is secreted by the seminal vesicles and associates with the plasma membrane of sperm upon ejaculation. The objective of this study was to demonstrate that PDC-109 promotes bull sperm binding to oviductal epithelium. PDC-109 was purified from bovine seminal plasma, and polyclonal antibodies were produced in rabbits. The antibodies detected PDC-109 on ejaculated sperm by indirect immunofluorescence and Western blots of extracts, but PDC-109 was not detected on epididymal sperm. When added to epididymal sperm, purified PDC-109 was absorbed onto the plasma membrane overlying the acrosome, as demonstrated by indirect immunofluorescence and by labeling sperm directly with fluorescein-conjugated PDC-109. When added to explants of oviductal epithelium, significantly fewer epididymal sperm than ejaculated sperm became bound. Addition of PDC-109 to epididymal sperm increased epithelial binding to the level observed for ejaculated sperm. In addition, binding of ejaculated sperm to oviductal epithelium was inhibited by addition of excess soluble PDC-109. Ejaculated sperm lost the ability to bind to oviductal epithelium after heparin-induced capacitation, but treatment with PDC-109 restored binding. These results demonstrate that PDC-109 enables sperm to bind to oviductal epithelium and plays a major role in formation of the bovine oviductal sperm reservoir.     

Sequestration of soil nitrogen as tannin-protein complexes may improve the competitive ability of sheep laurel (Kalmia angustifolia) relative to black spruce (Picea mariana)

The role of litter tannins in controlling soil nitrogen (N) cycling may explain the competitive ability of Kalmia relative to black spruce (Picea mariana), although this has not been demonstrated experimentally. Here, the protein-precipitation capacities of purified tannins and leaf extracts from Kalmia and black spruce were compared. The resistance to degradation of tannin-protein precipitates from both species were compared by monitoring carbon (C) and N dynamics in humus amended with protein, purified tannins or protein-tannin precipitates. The purity of the precipitates was verified using solid-state (13)C nuclear magnetic resonance (NMR) spectra. The ability of mycorrhizal fungi associated with both species to grow on media amended with tannin-protein complexes as the principal N source was also compared. The protein precipitation capacity of Kalmia tannins was superior to those of black spruce. Humus amended with protein increased both mineral and microbial N, whereas humus amended with tannin-protein precipitates increased dissolved organic N. Mycorrhizal fungi associated with Kalmia showed better growth than those associated with black spruce when N was provided as tannin-protein precipitates. These data suggest that Kalmia litter increases the amount of soil N sequestered as tannin-protein complexes, which may improve the competitive ability of Kalmia relative to black spruce by favouring N uptake by mycorrhizas associated with the former.     

An N-terminal deletion mutant of simian virus 40 (SV40) large T antigen oligomerizes incorrectly on SV40 DNA but retains the ability to bind to DNA polymerase alpha and replicate SV40 DNA in vitro.

A peptide encompassing the N-terminal 82 amino acids of simian virus 40 (SV40) large T antigen was previously shown to bind to the large subunit of DNA polymerase alpha-primase (I. Dornreiter, A. Höss, A. K. Arthur, and E. Fanning, EMBO J. 9:3329-3336, 1990). We report here that a mutant T antigen, T83-708, lacking residues 2 to 82 retained the ability to bind to DNA polymerase alpha-primase, implying that it carries a second binding site for DNA polymerase alpha-primase. The mutant protein also retained ATPase, helicase, and SV40 origin DNA-binding activity. However, its SV40 DNA replication activity in vitro was reduced compared with that of wild-type protein. The reduction in replication activity was accompanied by a lower DNA-binding affinity to SV40 origin sequences and aberrant oligomerization on viral origin DNA. Thus, the first 82 residues of SV40 T antigen are not strictly required for its interaction with DNA polymerase alpha-primase or for DNA replication function but may play a role in correct hexamer assembly and efficient DNA binding at the origin.