We have analyzed the functional domain structure of rat mammary glucosidase I, an enzyme involved in N-linked glycoprotein
processing, using biochemical and immunological approaches. The enzyme ...contains a high mannose type sugar chain that can be
cleaved by endo-beta-N-acetyl-D-glucosaminidase H without significantly affecting the catalytic activity. Based on trypsin
digestion pattern and the data on membrane topography, glucosidase I constitutes a single polypeptide chain of 85 kDa with
two contiguous domains: a membrane-bound domain that anchors the protein to the endoplasmic reticulum and a luminal domain.
A catalytically active 39-kDa domain could be released from membranes by limited proteolysis of saponin-permeabilized membranes
with trypsin. This domain appeared to contain the active site of the enzyme and had the ability to bind to glucosidase I-specific
affinity gel. Phase partitioning with Triton X-114 indicated the amphiphilic nature of the native enzyme, consistent with
its location as an integral membrane protein, whereas the 39-kDa fragment partitioned in the aqueous phase, a characteristic
of soluble polypeptide. These results indicate that glucosidase I is a transmembrane protein with a luminally oriented catalytic
domain. Such an orientation of the catalytic domain may facilitate the sequential processing of asparagine-linked oligosaccharide,
soon after its transfer en bloc by the oligosaccharyl transferase complex in the lumen of endoplasmic reticulum.
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.
Genome-wide assessment of genetic diversity has the potential to increase the ability to understand admixture, inbreeding, kinship and erosion of genetic diversity affecting both captive (
) and wild ...(
) populations of threatened species. The sable antelope (
), native to the savannah woodlands of sub-Saharan Africa, is a species that is being managed
in both public (zoo) and private (ranch) collections in the United States. Our objective was to develop whole genome sequence resources that will serve as a foundation for characterizing the genetic status of
populations of sable antelope relative to populations in the wild. Here we report the draft genome assembly of a male sable antelope, a member of the subfamily Hippotraginae (Bovidae, Cetartiodactyla, Mammalia). The 2.596 Gb draft genome consists of 136,528 contigs with an N50 of 45.5 Kbp and 16,927 scaffolds with an N50 of 4.59 Mbp.
annotation identified 18,828 protein-coding genes and repetitive sequences encompassing 46.97% of the genome. The discovery of single nucleotide variants (SNVs) was assisted by the re-sequencing of seven additional captive and wild individuals, representing two different subspecies, leading to the identification of 1,987,710 bi-allelic SNVs. Assembly of the mitochondrial genomes revealed that each individual was defined by a unique haplotype and these data were used to infer the mitochondrial gene tree relative to other hippotragine species. The sable antelope genome constitutes a valuable resource for assessing genome-wide diversity and evolutionary potential, thereby facilitating long-term conservation of this charismatic species.
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.
•The addition of Arnica montana (AM) to the IVM reduced cleavage and blastocyst rates.•The addition of ethanol to the IVM increases cleavage and blastocyst rates than AM.•During the heat stress (HS), the ethanol or AM increased embryo rate than control.•The Arnica montana in the presence of HS had a higher total cell number/blastocyst.
Sustaining viable populations of all wildlife species requires the maintenance of habitat, as well as an understanding of the behaviour and physiology of individual species. Despite substantial ...efforts, there are thousands of species threatened by extinction, often because of complex factors related to politics, social and environmental conditions and economic needs. When species become critically endangered, ex situ recovery programmes that include reproductive scientists are the usual first line of defence. Despite the potential of reproductive technologies for rapidly increasing numbers in such small populations, there are few examples of success. This is not the result of a failure on the part of the technologies per se, but rather is due to a lack of knowledge about the fundamental biology of the species in question, information essential for allowing reproductive technologies to be effective in the production of offspring. In addition, modern conservation concepts correctly emphasise the importance of maintaining heterozygosity to sustain genetic vigour, thereby limiting the practical usefulness of some procedures (such as nuclear transfer). However, because of the goal of maintaining all extant gene diversity and because, inevitably, many species are (or will become) 'critically endangered', it is necessary to explore every avenue for a potential contributory role. There are many 'emerging technologies' emanating from the study of livestock and laboratory animals. We predict that a subset of these may have application to the rescue of valuable genes from individual endangered species and eventually to the genetic management of entire populations or species. The present paper reviews the potential candidate techniques and their potential value (and limitations) to the study and conservation of rare wildlife species.
Of the 37 felid species, all but the domestic cat are classified as threatened with extinction in all or part of their native range. Additionally, the domestic cat is a valuable model for human ...biomedical research. Propagating some wild felids as well as domestic cat populations serving as human models is a major challenge primarily due to difficulties in transporting animals between facilities to ensure the pairing of genetically matched individuals, behavioral incompatibility between pairs and low fertility. Artificial insemination (AI) and in vitro fertilization/embryo transfer (IVF/ET) are powerful tools for helping manage rare populations. Developing successful assisted reproductive techniques requires knowledge of the female reproductive cycle and precise control of ovarian activity. Successful ovarian stimulation for AI and IVF/ET has been achieved in at least one-third of all cat species. However, sensitivity to a given gonadotropin treatment appears highly species-specific, and poor responses are common, particularly in felid species that exhibit spontaneous ovulations. Furthermore, current gonadotropin regimens have been demonstrated to perturb female reproductive function often leading to reduced fertility. Overall, ovarian response to exogenous hormonal stimulation has been highly variable, and pregnancy success after AI or IVF/ET remains low (<20%) in most species. Therefore, there is an immediate need to develop improved regimens that would allow more predictable ovarian responses in felids. We contend that recent research involving the use of progestins to control the ovary prior to gonadotropin stimulation shows promise for providing consistent ovarian stimulation in felids.
Glucosidase I initiates the processing of asparagine-linked glycoproteins by excising the distal alpha 1,2-linked glucosyl
residue from the Glc3Man9GlcNAc2 oligosaccharide, soon after its en bloc ...transfer from the lipid-linked donor to the nascent
polypeptide. 1-Deoxynojirimycin, an analog of D-glucose, is a potent competitive inhibitor of the enzyme. Sulfhydryl-seeking
reagents also strongly inhibit the enzyme, implying the involvement of an -SH group in its activity. To test this hypothesis,
glucosidase I was purified from the rat mammary gland and its active site was loaded with 1-deoxynojirimycin, to protect such
a group(s), while -SH groups on the remaining surface of the enzyme were blocked with N-ethylmaleimide or para-chloromercuriphenylsulfonic
acid. Deoxynojirimycin was removed by dialysis to expose the active site -SH group(s). This group(s) was then tagged with
3-(N-maleimidopropionyl)biocytin (MPB) and detected with 125I-streptavidin on Western blots. A series of experiments is presented
to show that indeed a critical -SH group(s) is located within the catalytic site of the enzyme. Additionally, the enzyme also
possesses one or more sulfhydryls and disulfide bonds in its primary structure. The experimental approach outlined here should
apply to identify reactive sulfhydryl groups in other catalytically active proteins.
Preserving genetic diversity of the critically endangered Addra gazelle (Nanger dama ruficollis) could be enhanced through the use of frozen-thawed sperm and artificial insemination. Our aim was to ...characterize Addra ejaculate traits and to assess the effects of cholesterol-loaded cyclodextrin (CLC) on sperm cryosurvival. Fresh ejaculates were treated with CLC (0, 0.5, 1.5, 3.0, and 6.0 mg/ml) prior to cryopreservation. All males produced spermic ejaculates with >75% sperm motility. The mean ± SEM seminal volume, sperm concentration, percent motility, forward progression, and percent morphologically normal spermatozoa were 3.2 ± 0.3 ml, 1.2 ± 0.3 × 109, 75.82 ± 2.7%, 3.2 ± 0.3 (0–5 scale; 5 = most progressive), and 57.12 ± 3.8%, respectively. More than 92% contained an intact acrosome. There was no effect of time or in vitro incubation on progression or acrosomal integrity on thawed samples (P > 0.05). Spermatozoa pre-treated with 0.5 mg/ml CLC retained higher (P < 0.05) motility post-thaw than aliquots treated with 0, 3.0, or 6.0 mg/ml of CLC. Spermatozoa pre-treated with 0.5, 1.5, or 3.0 mg/ml CLC exhibited greater viability than counterparts (P < 0.05). Sperm kinetics including beat cross frequency (BCF), average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL) did not differ among samples (P > 0.05). Linearity (LIN) and straightness (STR) were different among samples after thawing. Results demonstrate treatment with CLC (0.5 mg/ml) protects Addra spermatozoa from cryo-damage. Reported advances will facilitate establishment of a frozen repository and support the genetic management of this critically endangered north African desert antelope.
We studied the Persian onager (Equus hemionus onager), an endangered equid subspecies. The objective was to characterize endocrine patterns and ovarian follicular dynamics of females as well as ...seminal traits and sperm sensitivity to cryopreservation in males as a prerequisite to testing the feasibility of artificial insemination (AI). Urinary progesterone and estrogen metabolite profiles were determined by enzyme immunoassay in 11 females. Serial ultrasonography of ovarian activity was performed for 2 mo in a subset of four females. Females were seasonally polyestrous (June-November). Ovarian morphometry via ultrasonography and urinary progesterone profiles were more reflective of reproductive events than urinary estrogen patterns, and preovulatory follicle size was smaller than reported for other equid species. There was evidence for lactational suppression of estrus for up to 1.5 yr in nursing dams. Electroejaculation allowed recovery of highly motile sperm from 7, anesthetized males on 57% of occasions. Spermatozoa, including motility and acrosomal integrity, were resilient to freeze-thawing. Artificial insemination was successful in 2 of 3 females following detection of a dominant follicle and deslorelin administration, resulting in births of a healthy female and male foal by using fresh/chilled and frozen/thawed sperm, respectively.
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