The objective of this study was to identify factors that would allow the establishment of a serum-free culture system that could support follicular and oocyte growth, antrum formation, and estradiol-17beta (E(2)) production in preantral follicles of bovine ovaries. Large preantral follicles (145-170 micro m in diameter) were microsurgically dissected from ovaries, embedded in 0.15% type I collagen gels, and maintained in a serum-free medium for up to 13 days at 38.5 degrees C in 5% CO(2) in air. This culture environment allowed most preantral follicles to maintain a three-dimensional structure with the presence of a thecal layer and basement membrane surrounding the granulosa cells throughout the entire culture period. The effects of insulin, insulin-like growth factor (IGF)-I, IGF-II, FSH, and LH on preantral follicle growth were investigated in serum-free medium. Follicular diameters were significantly larger in the presence of insulin, IGF-I, IGF-II, or FSH after 13 days in culture. Oocyte diameters were also significantly larger in the presence of all hormones tested. The single addition of insulin, IGF-I, or FSH induced antrum formation between Days 7 and 13 of culture. Insulin progressively induced E(2) secretion by follicles after antrum formation, but IGF-I and FSH had no apparent effect. FSH and LH caused an increase in oocyte diameter in the presence of insulin. The addition of three hormones (insulin, FSH, and LH) initiated antrum formation and E(2) production earlier than insulin-containing medium alone. Furthermore, maximal E(2) secretion was maintained steadily between 7 and 13 days in this culture condition. From these results, we have demonstrated that insulin, FSH, and LH play substantial roles in the growth and development of bovine large preantral follicles in a serum-free medium.
Eggs were isolated from ovaries and oviducts of the golden hamster and the components of zonae pellucidae were examined using density gradient SDS-polyacrylamide electrophoresis. Zonae of ovarian eggs (ZP-OVA) had three major components corresponding to the so-called ZP-1, ZP-2, and ZP-3. Zonae of recently ovulated eggs collected from oviducts (ZP-OVI) had a 200-240 K component (ZP-O) in addition to the three components present in ZP-OVA. When ovarian and oviductal eggs were stained with FITC-conjugated B. simplicifolia-1 lectin (BS-1), which specifically binds to alpha-D-galactose- or alpha-N-acetyl-D-galactosamine-like terminal saccharide residues, ZP-OVI was intensely stained, while ZP-OVA was not. ZP-OVA gained the ability to bind to BS-1 after a brief treatment with oviduct extracts. These results suggest that biochemical properties of hamster zonae change after transport of eggs from ovary to the oviduct. The addition of the 200-240 K component of oviductal origin to preexisting zona components seems to be responsible for this change.
In the present study, we have isolated the cDNA for the mouse oviduct-specific glycoprotein (MOGP) by screening the mouse oviduct cDNA library with the bovine oviduct-specific glycoprotein (BOGP)-cDNA probe and by the 5' rapid amplification of the cDNA end (5'RACE). The total length of cDNA was determined to be 2525 base pairs (bp) by sequence analysis. The coding region contained 2163 bp translating to 721 amino acids. Based on comparisons with the N-terminal amino acid sequences of purified-BOGP and of hamster oviduct-specific glycoprotein (oviductin), it was inferred that the derived amino acid sequence contained a signal peptide region of 21 amino acids and a mature MOGP (core protein) region of 700 amino acids (76,515 daltons). It was also inferred that the mature MOGP contained three potential N-linked glycosylation sites and 24 possible O-linked glycosylation sites, and had the unique seven-residue repeat sequence (21 repeats) within the predicted sequence in the C-terminal side. The amino acid sequence of a portion of MOGP was highly homologous to that of BOGP (71% identity), baboon oviduct-specific glycoprotein (61% identity), and human oviduct-specific glycoprotein (77% identity). Significant homologies were also observed with two mammalian secretory proteins that were reported as a mammalian member of a chitinase protein family. Northern blot hybridization with a DIG-labeled probe indicated that a single message of 2.8 kb was present in total RNA prepared from oviductal tissue. In situ hybridization using MOGP-cDNA showed that a MOGP message was only detected in the oviductal epithelial cells. These results strongly suggest that a significant degree of homology exists among oviduct-specific glycoproteins of various mammalian species.
A specific 85-97-kDa (95-kDa) glycoprotein was found in bovine oviductal tissue and fluid during the follicular phase. In this study, a 95-kDa bovine oviductal glycoprotein (95-kDa BOGP) was purified by wheat germ agglutinin affinity and Mono-Q ion-exchange column chromatography. The first 29 NH2-terminal amino acid residues were determined by gas-phase microsequencing. A cDNA expression library prepared from poly(A)+ RNA isolated from bovine oviducts was screened with a monoclonal antibody to 95-kDa BOGP. A single positive clone containing a approximately 2-kb cDNA insert was isolated. The coding region contained 1612 bp translating to 537 amino acids. The derived amino acid sequence contained a partial signal sequence of 18 amino acids followed by 29 amino acids that were identical to the NH2-terminal amino acids determined by protein sequencing of purified 95-kDa BOGP. The amino acid sequence predicted a mature protein of 519 amino acids (57,684 daltons) containing one potential N-linked glycosylation site and five cysteines. Northern blot hybridization with a digoxigenin-labeled probe indicated that a single message of approximately 2.5 kb was present in oviductal RNA, and this message was detected in significantly greater amounts in oviductal RNA during the follicular phase than during the luteal phase. The amino acid sequence of a portion of 95-kDa BOGP was highly homologous (71% identity) to that of a baboon oviduct-specific glycoprotein.
In the cow, a specific glycoprotein--bovine oviduct-specific glycoprotein (BOGP)--is secreted by the epithelial cells of the oviduct at the follicular stage of the estrous cycle. In this study, we examined the effects of purified BOGP on the viability and motility of bovine spermatozoa in culture in vitro. Frozen-thawed bovine spermatozoa were incubated in modified Tyrode's solution (TALP) that contained purified BOGP (TALP-BOGP). In TALP-BOGP, both the viability and motility of bovine spermatozoa were more effectively maintained than in the control medium without any added protein. The increases in both the viability and motility of spermatozoa were dose-dependent. Spermatozoa were also incubated in TALP medium supplemented with bovine serum albumin, egg albumin, lactalbumin, or gastric mucin, and their viability and motility in these media were compared with that in TALP-BOGP. Both the viability and motility of spermatozoa were more effectively maintained in TALP-BOGP throughout a 12-hr incubation than in other media tested. An immunolabeling study demonstrated that a monoclonal antibody specific for BOGP reacted with the posterior region of the head, the middle portion, and the tail of spermatozoa that had been incubated with TALP-BOGP, suggesting that BOGP becomes specifically associated with particular regions of the spermatozoon. These results suggest that BOGP is a potent factor for maintenance of the viability and motility of sperm. On the basis of the present results, we also propose that BOGP may play an important role in sperm functions during the reproductive process.
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