The advent of in vitro fertilisation (IVF) and embryo transfer in the 1970s led to speculation about the potential value of these and other 'reproductive technologies' to conserving endangered ...species. So far, and for the most part, assisted breeding techniques that are routine in domesticated species are not easily adapted to wildlife. Species differences in reproductive form (anatomy/morphology) and function (mechanisms regulating reproductive success) limit the practical applicability for offspring production. Thus, the limiting factor is the lack of basic knowledge about thousands of unstudied species, the foundation that is essential to allowing reproduction to be enhanced and/or controlled. There now is excellent evidence that reproductive technologies are most useful as tools for studying how different species reproduce, especially defining novel and unique mechanisms. The present paper reviews the status and relevance of various reproductive technologies that are useful or have potential for wildlife. Modern examples of progress are provided indicating how these tools are being used to understand ways that wildlife species reproduce and, in some cases, how such knowledge has been used for successful assisted breeding, improved management and conservation.
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.
Most conventional spermatology research involves common mammalian species including livestock, laboratory animals and humans. Yet, there are more than 4500 mammalian species inhabiting the planet for ...which little is known about basic reproductive biology, including sperm characteristics and function. This information is important, not just as adjunct knowledge, but because the majority of these species are threatened with extinction, largely due to human-induced pressures. The field of conservation is changing rapidly, and global cooperation is emerging among a variety of wildlife enthusiasts, ranging from management authorities of nature reserves to curators of rare zoological collections. Conservation progress depends on systematic, multidisciplinary research first to answer basic questions, with new data then applied to endangered species management plans. The reproductive physiologist is a crucial component of this scheme. Reproduction is the essence of species survival, and enormous effort needs to be directed at these 'untraditional' research species, subspecies and populations. Spermatology research combined with simultaneous efforts in endocrinology, embryology and cryopreservation (among others) can lead to the successful application of assisted reproduction. Examples from this laboratory include an array of wild felid species and a rare cervid and mustelid. Obstacles to success are formidable, including unique species-specificities, diminished genetic diversity and a general lack of resources. Nonetheless, the field offers tremendous opportunities for generating unique knowledge of comparative interest and with conservation utility.
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.
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°C (no cooling; control), 2) cooled
to 5°C in a commercial refrigerator for 30 min (rapid cooling; â¼4°C/min), 3) placed in an ice slush at 0°C for 10 min (ultrarapid
cooling; â¼14°C/min), or 4) cooled to 0°C at 0.5°C/min in a programmable alcohol bath (slow cooling); and aliquots were removed
every 4°C. All samples then were warmed to 25°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°C. However, the proportion of spermatozoa
with intact acrosomes declined ( p < 0.05) after rapid cooling (â¼4°C/min) to 5°C (N, 65.6%; T, 27.5%) or ultrarapid cooling (â¼14°C/min) to 0°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°C/min) to 5°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°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.
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 CaCl
2), 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 μM) or lysophosphatidylcholine (LC; 100 μg/mL). Fresh sperm aliquots in TALP + 10 mM CaCl
2 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 CaCl
2 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 CaCl
2 evokes capacitation in some spermatozoa. However, Eld's deer spermatozoa appear remarkably resistant to conventional stimulators of capacitation and the AR.
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