The steroid metabolome of pregnancy, insights into the maintenance of pregnancy and evolution of reproductive traits.

Modes of mammalian reproduction are diverse and not always conserved among related species. Progesterone is universally required to supports pregnancy but sites of synthesis and metabolic pathways vary widely. The steroid metabolome of mid-to late gestation was characterized, focusing on 5α-reduced pregnanes in species representing the Perissodactyla, Cetartiodactyla and Carnivora using mass spectrometry. Metabolomes and steroidogenic enzyme ortholog sequences were used in heirarchial analyses. Steroid metabolite profiles were similar within orders, whales within cetartiodactyls for instance, but with notable exceptions such as rhinoceros clustering with goats, and tapirs with pigs. Steroidogenic enzyme sequence clustering reflected expected evolutionary relationships but once again with exceptions. Human sequences (expected outgroups) clustered with perissodactyl CYP11A1, CYP17A1 and SRD5A1 gene orthologues, forming outgroups only for HSD17B1 and SRD5A2. Spotted hyena CYP19A1 clustered within the Perissodactyla, between rhinoceros and equid orthologues, whereas CYP17A1 clustered within the Carnivora. This variability highlights the random adoption of divergent physiological strategies as pregnancy evolved among genetically similar species.

Impact of ethanol and heat stress-dependent effect of ultra-diluted Arnica montana 6 cH on in vitro embryo production in cattle.

This study evaluated the effect of adding ultra-diluted and dynamized Arnica montana 6 cH, and its vehicle (0.3% ethanol) to the in vitro maturation (IVM) medium, in the absence (experiment 1) or presence (experiment 2) of heat stress (HS), on bovine oocyte maturation and in vitro embryo production (IVEP). In experiment 1 (n = 902 cumulus oocyte complexes, COCs), the treatments were 1) IVM medium (Control treatment), 2) IVM medium + 0.3% ethanol, and 3) IVM medium + Arnica montana 6 cH. In experiment 2 (n = 1064 COCs), the treatments were 1) IVM medium without HS, 2) IVM medium under HS, 3) IVM medium + ethanol under HS, and 4) IVM medium + Arnica montana under HS. In the absence of HS (experiment 1), the addition of Arnica montana to the IVM medium had a deleterious effect on the IVEP (cleavage and blastocyst rates) and the total cell number/blastocysts. On the other hand, ethanol (0.3%) increased IVEP in relation to the Control and Arnica montana treatments. However, in the presence of HS during IVM (experiment 2), the addition of ethanol or Arnica montana increased IVEP when compared to the HS treatment alone, and the Arnica montana treatment resulted in greater total cell number/blastocysts compared to the other treatments. In conclusion, this study showed for the first time that the negative or positive effect of Arnica montana 6 cH on IVEP depends on the culture condition (i.e., absence or presence of HS during IVM). On the other hand, ethanol showed beneficial and consistent results on IVEP regardless of exposure to HS.

Single layer centrifugation of fresh dromedary camel semen improves sperm quality and in vitro fertilization capacity compared with simple sperm washing.

Single layer centrifugation (SLC) through a colloid is a tool for selecting viable mammalian spermatozoa but has not been used previously for fresh dromedary camel sperm. Semen from six camels (2 ejaculates/male) was diluted 1:5 (v:v) or 1:10 (v:v) in a Tris-citrate-fructose buffer for mechanical liquefaction by gentle pipetting. Following liquefaction, semen was processed either by SLC or by centrifugation without a colloid (control). Total and progressive motilities, CASA kinematics, vitality and acrosome integrity (eosin-nigrosin) and plasma membrane integrity (Hypo-osmotic swelling test; HOST), and fertilizing ability in a heterologous assay (zona-free goat oocytes) were evaluated. Both total (p = .003) and progressive motilities (p = .003) were higher in SLC-processed than in control semen samples, irrespective of dilution. Positive HOST values increased when using colloid in 1:5 (p = .001) and 1:10 dilution (p = .010). Colloid-selected sperm had higher penetration rates than controls (p < .001 and p = .02 for 1:5 and 1:10 dilutions, respectively). However, only the SLC sperm at 1:5 dilution showed higher percentages of pronuclear formation (p = .02) than controls. Dilution effect was only significant for total motility before in vitro fertilization, with higher values for the 1:5 dilution (p = .033). The recovery rates of motile sperm between dilutions were similar (26.1% vs 35.4%; p = .226). In conclusion, SLC is a promising tool for selecting functional dromedary camel sperm and warrants more research.

Analysis of Sertoli cell efficiency allows the differentiation between two fundamentally different forms of feline teratospermia.

Teratospermia is a common phenomenon within felid species and has been attributed to reduction in genetic diversity. Testes from teratospermic domestic cats show enhanced spermatogenesis accompanied by remarkably reduced germ cell apoptosis. In the present study we investigated whether free-range teratospermic tom cats exhibit a similar testicular phenotype as proven permanently teratospermic males. Randomly collected teratospermic cats were compared with normal (normospermic; >60% morphologically normal sperm per ejaculate) and a well-characterized population of permanently teratospermic domestic cats, with respect to their spermatogenic potential. Histomorphologic assessment of testes from randomly collected teratospermic cats revealed no differences compared with normospermic donors. These two groups, however, were both different from permanently teratospermic cats, which exhibit fewer Sertoli cells and increased numbers of round spermatids per tubule cross-section resulting in a remarkably increased Sertoli cell efficiency (ratio of round spermatids to Sertoli cells). In conclusion, we can distinguish at least two fundamentally different forms of feline teratospermia. One subtype, found in most of the randomly collected tom cats, but not associated with altered quantitative spermatogenic parameters. Another subtype, found in all permanently teratospermic felids, is manifested by an impairment of Sertoli cell efficiency. We suggest that spermatogenic output should be analyzed before using random source domestic cats to study the phenomenon of teratospermia.

Molecular markers of spermatogonial stem cells in the domestic cat.

Spermatogonial stem cells (SSCs) represent an exciting new avenue for assisted reproduction in endangered and genetically valuable species. Before this technology can be applied to wildlife, species-specific markers are required to evaluate SSC enrichment strategies and monitor subsequent in vitro culture. This study was designed to evaluate six conserved SSC markers (THY1, GPR125, GFRalpha1, PLZF, UCHL1 and OCT4) in the cat. Testes from three juveniles and three adults were obtained following routine castrations and processed for mRNA extraction. RT-PCR of whole testis and cell suspensions enriched for SSCs by differential plating confirmed that all six SSC markers are expressed in both the whole testis and SSC-enriched cell fractions. The expression of all six putative SSC marker genes in the cat testis suggests conservation of SSC markers, and perhaps self-renewal mechanisms, in felids.

The competence of germinal vesicle oocytes is unrelated to nuclear chromatin configuration and strictly depends on cytoplasmic quantity and quality in the cat model.

BACKGROUND: Chromatin configuration of the germinal vesicle (GV) and quality of the cytoplasm are critical factors in achieving oocyte meiotic and developmental capacity during folliculogenesis. Besides gaining new insights into the timing and cellular mechanisms associated with the acquisition and regulation of GV oocyte competence, the domestic cat model was used to examine (i) the relation between GV chromatin configuration and oocyte functionality during folliculogenesis and (ii) the role of the cytoplasmic environment on the GV competence and stability. METHODS: Structural and functional properties of GV oocytes were characterized after isolation from different follicle stages of non-stimulated cat ovaries. GV transfers, artificial chromatin compaction and oocyte vitrification were used to demonstrate the respective roles of GV and cytoplasm on the oocyte functionality. RESULTS: GVs acquired the intrinsic capability to resume meiosis during the pre-antral follicle stage, whereas the capacity to support embryo development occurred while the antrum started to form. Chromatin configuration of the GV did not undergo extensive modification during the acquisition of competence or during the arrest of transcriptional activity at the large antral follicle stage. However, the quality and quantity of the cytoplasm regulated and enhanced GV functionality. This finding also held for GVs transferred from incompetent or subpar oocytes into the cytoplasm of good quality oocytes or when chromatin was artificially modified or vitrified. CONCLUSIONS: The cat model provides a new insight into GV oocyte structure and function during folliculogenesis while challenging current concepts about oocyte quality criteria based on the GV morphology. This suggests alternative evaluative approaches for oocytes from other species too, including humans. Cat GVs also appear competent at an early follicle stage and are resilient to perturbations which designate this organelle as an attractive target for developing novel fertility preservation tactics.

Oocyte quality and estradiol supplementation affect in vitro maturation success in the white-tailed deer (Odocoileus virginianus).

White-tailed deer oocyte biology is not well documented. The objective of this study was to determine (1) the influence of estradiol (E(2)) supplementation on meiotic resumption and the ability to "rescue" poorer quality (lower grade) oocytes and (2) the kinetics of oocyte nuclear maturation in vitro in the white-tailed deer. In Experiment 1, immature oocytes harvested during hunting-culling operations were cultured for 24h in the presence or absence of E(2). Incubation in 1mug/mL E(2) promoted nuclear maturation (to telophase I, TI; or to metaphase II, MII) in a higher proportion of Grade 1 oocytes ( approximately 77%; P<0.05) compared with that in Grade 2 or Grade 3 counterparts ( approximately 51%). For Grades 2 and 3 oocytes, there was no advantage (P>0.05) for E(2) supplementation in reaching TI/MII. In Experiment 2, Grade 1 oocytes were cultured in the presence of E(2) and nuclear status evaluated at 0, 3, 6, 12, and 24h of in vitro incubation. At 0h,>70% of oocytes already had undergone germinal vesicle breakdown. After 12h, approximately 70% of oocytes had reached metaphase I of nuclear maturation, with approximately 75% achieving TI/MII by 24h in vitro. In summary, adding E(2) to an in vitro maturation (IVM) culture system for white-tailed deer was advantageous, but only for the highest quality oocytes, with approximately 75% achieving nuclear maturation. In contrast, E(2) supplement did not benefit lower-grade oocytes, half of which will reach MII, with the other half failing. Under the described culture conditions, good-quality white-tailed deer oocytes achieve nuclear maturation over a time duration comparable with that reported in other ungulates.

Follicular morphology, oocyte diameter and localisation of fibroblast growth factors in the domestic dog ovary.

Remarkably little is known about folliculogenesis in the dog. Objectives were to characterise (1) changes in follicle/oocyte diameter and granulosa cell number and (2) localisation of fibroblast growth factor (FGF)-2 and FGF-7 during dog ovarian follicle development. Fourteen ovarian pairs were excised and processed for histological evaluation. Two to four serial sections/bitch were stained with hematoxylin, and follicle/oocyte diameters and granulosa cell number were determined at each developmental stage. Mean follicle and oocyte size were compared among stages by one-way analysis of variance. Relationships between follicle and oocyte size and granulosa cell number were determined using correlation and regression analysis, respectively. Another eight serial sections/bitch were processed for immunostaining to determine FGF-2 and FGF-7 localisation. Primordial and primary follicles were similar in size, but smaller than the progressively increasing (p < 0.05) diameter of the later stages. Oocyte diameter gradually increased (p < 0.05) among oocytes derived from primordial, primary, secondary and early antral follicles with no difference (p > 0.05) thereafter. Oocyte size and granulosa cell number increased (p < 0.01) with follicular diameter. Except during anoestrus, FGF-2 occurred in oocytes and granulosa cells of primordial to secondary follicles. In adult bitches, FGF-7 was localised in granulosa cells of primary and secondary follicles and also occurred in the theca layer of antral follicles during prooestrus and oestrus. In summary, folliculogenesis in the domestic dog occurs in two phases: pre-antral phase characterised by increasing follicle size in association with oocyte growth and granulosa cell proliferation and antral phase linked with marked granulosa cell proliferation and accumulation of antral cavity fluid. Finally, the temporal localisation pattern of FGF-2 implies its role in follicular activation, whereas FGF-7 activities appear related to later folliculogenesis.

In vitro compaction of germinal vesicle chromatin is beneficial to survival of vitrified cat oocytes.

The immature cat oocyte contains a large-sized germinal vesicle (GV) with decondensed chromatin that is highly susceptible to cryo-damage. The aim of the study was to explore an alternative to conventional cryopreservation by examining the influence of GV chromatin compaction using resveratrol (Res) exposure (a histone deacetylase enhancer) on oocyte survival during vitrification. In Experiment 1, denuded oocytes were exposed to 0, 0.5, 1.0 or 1.5 mmol/l Res for 1.5 h and then evaluated for chromatin structure or cultured to assess oocyte meiotic and developmental competence in vitro. Exposure to 1.0 or 1.5 mmol/l Res induced complete GV chromatin deacetylation and the most significant compaction. Compared to other treatments, the 1.5 mmol/l Res concentration compromised the oocyte ability to achieve metaphase II (MII) or to form a blastocyst. In Experiment 2, denuded oocytes were exposed to Res as in Experiment 1 and cultured in vitro either directly (fresh) or after vitrification. Both oocyte types then were assessed for meiotic competence, fertilizability and ability to form embryos. Vitrification exerted an overall negative influence on oocyte meiotic and developmental competence. However, ability to reach MII, achieve early first cleavage, and develop to an advanced embryo stage (8-16 cells) was improved in vitrified oocytes previously exposed to 1.0 mmol/l Res compared to all counterpart treatments. In summary, results reveal that transient epigenetic modifications associated with GV chromatin compaction induced by Res is fully reversible and beneficial to oocyte survival during vitrification. This approach has allowed the production of the first cat embryos from vitrified immature oocytes.

Localization of oestrogen receptors in the epididymis during sexual maturation of the domestic cat.

Oestrogens are involved in regulation of spermatogenesis and sperm maturation and are essential for male fertility. To study the role of oestrogens on epididymal function in the domestic cat, we analyzed the localization patterns of oestrogen receptors (ERs) within the epididymis of juvenile, pubertal and adults using immunohistochemistry. Cat epididymal tissues obtained during routine castrations were fixed in chilled Bouin's solution and processed for immunohistochemistry with ER-specific antibodies. For a certain receptor type, ER localization was influenced by donor age. In the juvenile epididymis, ERalpha was localized in the nuclei of epithelial cells of efferent ducts and undifferentiated epithelium of the ductus epididymis. During puberty, ERalpha localization in the undifferentiated epithelium of the epididymis shifted from the nuclei to the cytoplasm and plasma membrane. Oestrogen receptor-alpha level was highest in the pubertal and adult epididymis, especially within the cytoplasm and in plasma membranes of caput epithelial cells. This finding was suggestive of a role in fluid reabsorption within the efferent ducts and the epididymis. In corpus and cauda regions, ERalpha was less abundant, suggesting a minor role for oestrogens in sperm storage areas. Interestingly, localization of ERbeta was neither influenced by age nor location within the epididymis and was ubiquitous throughout. Results demonstrate that oestrogen actions within the epididymis may be predominantly mediated through ERalpha during sexual maturation in the domestic cat.

Influence of cooling rate on the ability of frozen-thawed sperm to bind to heterologous zona pellucida, as assessed by competitive in vitro binding assays in the ocelot (Leopardus pardalis) and tigrina (Leopardus tigrinus).

We evaluated the influence of two cooling rates (from 25 to 5 degrees C) on post-thaw function of frozen sperm in ocelots (Leopardus pardalis; n=3 males) and tigrinas (Leopardus tigrinus; n=4 males). Seven normospermic (>70% normal sperm) electroejaculates from each species were diluted with a 4% glycerol freezing medium, divided into two aliquots, and assigned to one of two cooling rates: fast or slow (0.7 or 0.16 degrees C/min, respectively). Sperm motility index (SMI) and percentage of sperm with an intact acrosome were assessed before freezing and after thawing, and the ability of sperm to bind to the zona pellucida of IVM domestic cat oocytes were assessed in a competitive in vitro sperm-binding assay. Regardless of the cooling rate, frozen-thawed sperm from both species exhibited a SMI of 50; approximately 20 and approximately 32% of post-thaw sperm had an intact acrosome in ocelots and tigrinas, respectively (P<0.05). The mean (+/-S.E.M.) number of sperm bound per oocyte was higher for fast-cooled (8.5+/-1.3) than slow-cooled (2.5+/-0.3; P<0.01) ocelot sperm. In contrast, more tigrina sperm bound to domestic cat oocytes when cooled slowly versus quickly (5.8+/-0.9 versus 2.7+/-0.4, P<0.05). In conclusion, cryopreservation decreased sperm function in both species, and the oocyte-binding assay was the most efficient method to detect functional differences in post-thaw sperm.

Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season.

The domestic cat experiences circannual variations in ovarian activity and intrafollicular oocyte quality. One result is poor nuclear and cytoplasmic maturation during in vitro maturation (IVM) conducted during the annual non-breeding season (July through November). In an attempt to overcome this seasonal phenomenon immature oocytes were collected from July through November and cultured in a conventional IVM medium (IVM1) or in IVM1 supplemented with different FSH concentrations and antioxidant (ascorbic acid or cysteine). Nuclear status of oocytes was assessed after IVM or IVF. Embryo stage and blastocyst quality were evaluated after 7 days of in vitro culture. Although the addition of antioxidant alone had no effect, the presence of 10 microg FSH ml(-1) improved nuclear maturation (75.4+/-4.1% versus 48.7+/-8.8% in IVM1; P<0.05) and fertilization success (47.9+/-3.2% versus 35.0+/-5.1% in IVM1; P<0.05). Furthermore, developmental competence of fertilized oocytes was enhanced (P<0.05) only in the presence of ascorbic acid (30.6+/-6.7%) or cysteine (33.6+/-5.1%) compared with IVM1 (8.1+/-8.8%). Consequently, blastocyst yield (17% of total oocytes cultured) was highest when oocytes were matured in medium containing higher FSH concentration and antioxidants. The results of this study demonstrate that meiotic and developmental competences are inherent to the immature cat oocyte collected during the non-breeding season. However, appropriate mechanisms (perhaps seasonal variation in FSH receptors or lack of antioxidant capacity of the cumulus-oocyte complex) are inadequate during this period of gonadal quiescence. Regardless, this compromised oocyte function during the non-breeding season can be overridden by altering in vitro culture conditions to include supplemental FSH and antioxidant.

Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation.

Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.

Sperm capacitation in vitro in the eld's deer.

Sperm capacitation was examined in the endangered Eld's deer (Cervus eldi thamin). Sperm motility and viability (percentage of sperm cells with intact membranes) were assessed in vitro over time after attempting to induce capacitation in TALP alone and TALP supplemented with calcium (10 mM CaCl2), dibutyryl cAMP (1 mM dbcAMP), or fetal calf serum (20% FCS). Sperm aliquots were evaluated at 0, 3, 6, 9, and 12 h for motility, viability, and ability to acrosome react after exposure to calcium ionophore (A23187, CI; 10 microM) or lysophosphatidylcholine (LC; 100 microg/mL). Fresh sperm aliquots in TALP + 10 mM CaCl2 exposed to CI had fewer (P < 0.05) intact acrosomes than the TALP control (TALP alone) or dbcAMP and FCS treatments after 9 h. Mean (+/- SEM) percentage of intact acrosomes of spermatozoa incubated in medium with increased CaCl2 declined (P < 0.05) from 80.2 +/- 2.6% (0 h) to 49.7 +/- 7.3% after prolonged incubation (9 h). The proportion of capacitated fresh spermatozoa was not influenced by LC treatment. Capacitation was not induced (P > 0.05) by any of the presumptive sperm capacitators after freeze-thawing. Likewise, neither CI nor LC induced the acrosome reaction (AR) in these spermatozoa, suggesting that the freeze-thawing process may have caused membrane damage. Results revealed that the supplementation of medium with CaCl2 evokes capacitation in some spermatozoa. However, Eld's deer spermatozoa appear remarkably resistant to conventional stimulators of capacitation and the AR.

The phenomenon and significance of teratospermia in felids.

The common domestic cat is an important research model for endangered felids, as well as for studying genetic dysfunctions, infectious diseases and infertility in humans. Especially significant is the trait of teratospermia (ejaculation of < 40% morphologically normal spermatozoa) that commonly occurs in about 70% of the felid species or subspecies studied to date. Teratospermia, discovered more than two decades ago in the cheetah, is important: (i) for understanding the significance of sperm form and function; and (ii) because this condition is common in human males. It is apparent from IVF that deformed spermatozoa from teratospermic felids do not fertilize oocytes. However, the inability of spermatozoa from teratospermic males to bind, penetrate and decondense in the cytoplasm of the oocyte is not limited to malformed cells alone. Normal shaped spermatozoa from teratospermic males have reduced functional capacity. IVF results have consistently revealed a direct correlation between teratospermia and compromised sperm function across felid species and populations. The most significant differences between normospermic (> 60% normal spermatozoa per ejaculate) and teratospermic felids include: (i) the time required for sperm capacitation and the acrosome reaction to occur in vitro; (ii) culture media requirements for capacitation in vitro; (iii) phosphorylation patterns of tyrosine residues on sperm membrane proteins during capacitation; (iv) susceptibility to chilling-induced sperm membrane damage; (v) sensitivity to osmotic stress; (vi) stability of sperm DNA; (vii) sperm protamine composition; and (viii) fertilizing ability after intracytoplasmic sperm injection. In conclusion, (i) the felids (including wild species) are valuable for studying the functional significance of both pleiomorphic and normally formed spermatozoa from teratospermic donors, and (ii) the impact of teratospermia is expressed at both macrocellular and subcellular levels.

Osmotic effects on feline spermatozoa from normospermic versus teratospermic donors.

Effects of osmolality stresses on the sperm of normospermic (>60% normal sperm/ejaculate) versus teratospermic (<40% normal sperm) domestic cats and the normospermic leopard cat and the teratospermic clouded leopard were studied. Spermatozoa were exposed to various anisotonic solutions in a single step or returned to near isotonic conditions in a single step after exposure to anisotonic solutions. The percentage of sperm motility was measured subjectively, and dual fluorescent stains were used to assess membrane integrity by flow cytometry. The percentage of sperm motility declined (P < 0.05) in domestic cat sperm exposed to osmolalities <200 and >450 mOsm. Spermatozoa from all felines underwent marked (P < 0.05) membrane disruption following a hypotonic stress, but sperm from teratospermic donors experienced greater (P < 0.05) membrane disruption in response to decreased osmolality. While feline spermatozoa appeared to be highly resistant to hypertonic (600, 1200, and 2400 mOsm) conditions, with >85% of the cells maintaining intact membranes, severe membrane disruption occurred when cells were returned to isotonicity in a single step. There was no difference (P > 0.05) between a 1- and 5-min exposure to various anisotonic solutions. Similarly, sperm from normospermic and teratospermic domestic cats responded identically after exposure to ionic or nonionic solute. Results demonstrate that: (1) spermatozoa from teratospermic males are more vulnerable to a hypotonic stress than sperm from normospermic counterparts; (2) in response to small deviations in osmolality, feline sperm experience a more rapid decline in motility than membrane integrity; and (3) an abrupt return to isotonicity after a hypertonic stress causes extensive sperm membrane damage regardless of ejaculate quality.

Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro.

Current methods for detecting complete oocyte maturation and developmental competence are inadequate. The objectives of this study were to (1) examine the relationship between cat oocyte energy metabolism and development in vitro after fertilization and (2) determine if cumulus cell metabolism could be used to predict development of individual oocytes after fertilization in vitro. The hanging drop method was used to assess metabolism of three different types of cat oocytes: immature (IMO), in vitro matured (IVM), and in vivo matured (IVOM). Stage of oocyte nuclear maturation or developmental competence was assessed after metabolic analysis. Glycolysis and oxidation of glucose, glutamine, palmitate, and lactate increased with the resumption of oocyte meiotic maturation (P<0.05). Pyruvate was the preferred substrate, but uptake was not linked to maturation. IVM oocytes had impaired glucose and palmitate metabolism compared to IVOM oocytes (P<0.05). Oocyte glycolytic activity and oocyte glucose oxidation correlated well with embryo development after insemination in vitro (P<0.05). Furthermore, oocytes that had similar glucose metabolism and that were grouped together for culture on this basis had higher (P<0.05) overall rates of development than oocytes grouped randomly. There was no correlation (P>0.05) between cumulus cell metabolism and individual oocyte development after in vitro fertilization. The data reveal that energy metabolism is linked to oocyte maturation in the cat and that glucose metabolic activity can indicate those oocytes most likely to fertilize and develop in vitro. Measuring cumulus cell metabolism does not accurately predict individual oocyte development after insemination in vitro.

Sensitivity of domestic cat (Felis catus) sperm from normospermic versus teratospermic donors to cold-induced acrosomal damage.

Freeze-thawing cat sperm in cryoprotectant results in extensive membrane damage. To determine whether cooling alone influences sperm structure and viability, we compared the effect of cooling rate on sperm from normospermic (N; > 60% normal sperm per ejaculate) and teratospermic (T; < 40% normal sperm per ejaculate) domestic cats. Electroejaculates were divided into raw or washed (Ham's F-10 + 5% fetal calf serum) aliquots, with the latter resuspended in Ham's F-10 medium or Platz Diluent Variant Filtered without glycerol (20% egg yolk, 11% lactose). Aliquots were 1) maintained at 25 degrees C (no cooling; control), 2) cooled to 5 degrees C in a commercial refrigerator for 30 min (rapid cooling; approximately 4 degrees C/min), 3) placed in an ice slush at 0 degrees C for 10 min (ultrarapid cooling; approximately 14 degrees C/min), or 4) cooled to 0 degrees C at 0.5 degrees C/min in a programmable alcohol bath (slow cooling); and aliquots were removed every 4 degrees C. All samples then were warmed to 25 degrees C and evaluated for percentage sperm motility and the proportion of intact acrosomes using a fluorescein-conjugated peanut agglutinin stain. In both cat populations, sperm percentage motility remained unaffected (p > 0.05) immediately after exposure to low temperatures and after warming to 25 degrees C. However, the proportion of spermatozoa with intact acrosomes declined (p < 0.05) after rapid cooling ( approximately 4 degrees C/min) to 5 degrees C (N, 65.6%; T, 27.5%) or ultrarapid cooling ( approximately 14 degrees C/min) to 0 degrees C (N, 62.1%; T, 23.0%) in comparison to the control value (N, 81.5%; T, 77.5%). Transmission electron microscopy of cooled sperm revealed extensive damage to acrosomal membranes. In contrast, slow cooling (0.5 degrees C/min) to 5 degrees C maintained (p > 0.05) a high proportion of spermatozoa with intact acrosomes (N, 75.5%; T, 68.3%), which also remained similar (p > 0.05) between cat populations (N, 64.7%; T, 56.8%) through continued cooling to 0 degrees C. Results demonstrate that 1) rapid cooling of domestic cat sperm induces significant acrosomal damage without altering sperm motility, 2) spermatozoa from teratospermic males are more susceptible to cold-induced acrosomal damage than normospermic counterparts, and 3) reducing the rate of initial cooling markedly decreases sperm structural damage.

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

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.

Inhibition of domestic cat spermatozoa acrosome reaction and zona pellucida penetration by tyrosine kinase inhibitors.

Spermatozoa from teratospermic domestic cats (> 60% morphologically abnormal spermatozoa per ejaculate) consistently exhibit lower levels of oocyte penetration in vitro than their normospermic (< 40% abnormal spermatozoa per ejaculate) counterparts. This could be caused by structural abnormalities or intracellular defects resulting in disruption of normal cellular functions. Spermatozoa from teratospermic cats also are compromised in the ability to capacitate and undergo the acrosome reaction (AR) in vitro. Further, we recently identified two tyrosine phosphorylated proteins (95- and 160-kDa) localized over the acrosome region in domestic cat spermatozoa. Phosphorylation of these proteins is reduced in teratospermic compared with normospermic ejaculates. To begin to understand the relationship between tyrosine phosphorylation and sperm function, we examined the effects of two protein tyrosine kinase inhibitors (tyrphostin RG-50864 and genistein) on (1) sperm motility; (2) protein tyrosine phosphorylation; (3) the ionophore A23187-induced AR; (4) the spontaneous and zona pellucida (ZP)-induced AR, and (5) the ability of spermatozoa from normospermic cats to penetrate conspecific ZP-intact oocytes. Over a wide range of concentrations, neither inhibitor affected sperm percentage motility during incubation (P > 0.05). Preincubation with either inhibitor reduced tyrosine phosphorylation of both (95- and 160-kDa) sperm proteins. Although both inhibitors blocked the ZP-induced AR, neither influenced the spontaneous AR nor the A23187-induced AR, suggesting that tyrosine phosphorylation may be involved in physiologic AR. No differences (P > 0.05) were observed in the ability of control or inhibitor-treated spermatozoa to bind to or penetrate the outer ZP layer. However, percentages of oocytes with treated spermatozoa in the inner ZP (tyrphostin, 8.7%; genistein, 20.4%) and perivitelline space (tyrphostin, 0%; genistein, 2.3%) were less (P < 0.001) than untreated controls (inner ZP, 62.7%; perivitelline space, 10.2%). These results (1) demonstrate that ZP-induced acrosomal exocytosis in domestic cat spermatozoa is regulated via a tyrosine kinase-dependent pathway and (2) suggest that defects in these signaling pathways may represent one of the causes for compromised sperm function in teratospermic males.