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.
Contents
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.
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.
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.
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.
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.
Teratozoospermia (ejaculation of <40% morphologically normal sperm) commonly occurs within the Felidae, including certain
domestic cats, but the cellular and molecular mechanisms that give rise to ...this phenomenon remain unknown. This study quantified
spermatogenesis to identify differential dysfunctions in teratospermic versus normospermic (>60% normal sperm/ejaculate) domestic
cats. Sperm used were from electroejaculates and cauda epididymides. Testes from 10 normo- and 10 teratospermic males were
obtained by castration and then evaluated by histomorphometry, flow cytometry, and testicular testosterone enzyme immunoassay.
Some morphometric traits (tubular diameter, epithelium height, interstitial area, number of Leydig cells, and blood vessels
per cross-section) as well as testicular testosterone concentrations were similar between groups, but testicular volume was
greater in teratospermic males. Stage frequencies differed also between both cat populations, suggesting possible dysfunctions
in spermiation. Quantification of cell populations in most frequent stages revealed more spermatogenic cells and fewer Sertoli
cells per tubule cross-section as well as per tissue unit in teratospermic donors. Hence, the ratio of spermatogenic cells
per Sertoli cell was elevated in the teratospermic cat. DNA flow cytometry confirmed higher total spermatogenic and meiotic
transformations in teratospermic males. In summary, compared with normospermic counterparts, teratospermic cats have a higher
sperm output achieved by more sperm-producing tissue, more germ cells per Sertoli cell, and reduced germ cell loss during
spermatogenesis. Gains in sperm quantity are produced at the expense of sperm quality.
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.
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 24
h in the presence or absence of E
2. Incubation in 1
μg/mL E
2 promoted nuclear maturation (to telophase I, TI; or to metaphase II, MII) in a higher proportion of Grade 1 oocytes (∼77%; P
<
0.05) compared with that in Grade 2 or Grade 3 counterparts (∼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 24
h of in vitro incubation. At 0
h,
>
70% of oocytes already had undergone germinal vesicle breakdown. After 12
h, ∼70% of oocytes had reached metaphase I of nuclear maturation, with ∼75% achieving TI/MII by 24
h 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 ∼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.
Contents
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.
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