A repeatable procedure for fertilization of bovine ova in vitro is described. Oocytes were recovered from ovarian follicles or from oviducts near the time of ovulation following treatment of donors with pregnant mare's serum gonadotropin (PMSG) and prostaglandin F2 alpha (PGF2 alpha). For in vitro capacitation semen was incubated, then high ionic strength treated and subsequently incubated in defined medium prior to insemination of oocytes. In one experiment frozen bull semen was successfully used. In experiments with 4 bulls (B, C, D, F), 34 (43.6%) of 78 ova and 13 (19.7%) of 66 follicular oocytes were fertilized in vitro. In the last series (spermatozoa from Bull F) the fertilization of 22 (62.9%) of 35 tubal ova was achieved. In vitro development proceeded to the 8-cell stage. No fertilization in vitro followed use of one male (Bull E), even though his spermatozoa could penetrate zona-free hamster ova in vitro, and higher than usual bacterial contamination of his semen was implicated as the probable cause. Findings suggested vigorous progressive sperm motility and acrosome integrity to be important features of good sperm samples. In one experiment a 4-cell stage embryo was transferred with the result that the recipient gave birth to a normal bull calf on June 9, 1981. The first calf resulting from in vitro fertilization has been found to be completely normal.

Published reports indicate that in several mammalian species the oviduct synthesizes and secretes specific glycoproteins which are components of the luminal fluids at the time of ovulation and fertilization. The present study characterized the secretory glycoproteins synthesized by the bovine oviduct at estrus. Oviducts obtained from four crossbred cows in estrus were flushed with saline, and segments of the ampullary and isthmic regions were fixed for immunocytochemical analyses. The remainder of the tissue was subjected to explant culture for 24 h in medium containing either 3H-leucine or 3H-glucosamine. Analysis of culture media by one- and two-dimensional SDS-PAGE followed by fluorography indicated that both the ampullary and isthmic regions synthesized a major class of Mr 97,000 glycoproteins with isoelectric points ranging from 5.5 to 8.1. A polyclonal antibody was generated to the glycoproteins after their isolation by gel filtration followed by electrophoretic separation. Western blot analysis of oviductal culture media indicated that the antisera cross-reacted with a doublet at Mr 97,000 and to a lesser extent with two additional bands at Mr greater than 200,000. Immunoreactive antigens were not identified in serum or in culture media of ovary, uterus, and nonreproductive tract tissues. The Mr 97,000 glycoproteins were present in oviductal flushings obtained from cows in estrus. They were also detected to a lesser degree in oviductal flushings obtained from cows at Days 5, 10, 15, and 18 of the estrous cycle, with the least amount of immunoreactivity being observed in Day 10 samples.(ABSTRACT TRUNCATED AT 250 WORDS)

The estrogen-dominated baboon oviductal epithelium synthesizes and secretes a family of oviduct-specific glycoproteins. The objective of this study was to determine if these glycoproteins become associated with ova and early embryos. Ovarian and oviductal eggs obtained from superovulated baboons 72 h post-hCG were subjected to an indirect immunofluorescent assay that used a polyclonal antibody prepared toward the baboon oviduct-specific glycoproteins. Oviductal ova as well as 2-cell and 4-cell embryos showed intense, specific fluorescence within their zonae pellucidae. Ovarian ova did not exhibit fluorescence. Oviductal eggs were also fixed and processed for peroxidase-antiperoxidase immunocytochemistry and colloidal gold immunoelectron microscopy to confirm the immunofluorescent data and to determine the subcellular distribution of the antigens. Oviductal ova as well as 2-cell and 3-cell embryos exhibited immunolabeling localized within the zona. Gold particles were distributed uniformly throughout the width of the zona. Occasional groupings of gold particles were observed within the zona. Also, in most eggs, immunoreactivity was observed associated with flocculent material in the perivitelline space as well as the vitelline membrane. Furthermore, immunogold labeling above background level was noted in the cytoplasm of the eggs, particularly in the blastomeres of 3-cell embryos. Collectively, these results indicate that baboon estrogen-dependent oviductal secretory glycoproteins become intimately associated with oviductal ova and with embryos.

Oviducts were obtained from a series of cycling and ovariectomized steroid-treated baboons. The lining epithelium of the ampulla and isthmus was analyzed by light and electron microscopy. Both morphological and cytomorphometric analyses revealed that the morphological and functional state of the oviductal epithelium in the baboon is controlled by the ovarian steroids. Additionally, a clear cephalocaudal steroid-responsive gradient was observed when the data from the ampulla and isthmus of the same animal were compared. Within the ampulla, estradiol induced hypertrophy, hyperplasia, ciliogenesis, and secretory activity, whereas adding progesterone to the treatment regimen (+/- estradiol) resulted in atrophy, deciliation, apoptosis, and loss of the secretory activity. These cyclic processes were less evident in the isthmus. We also used an indirect electron microscopic immunogold technique and a previously characterized polyclonal antibody to determine the localization of oviduct-specific glycoproteins. These glycoproteins were present in every secretory granule observed, regardless of oviduct region, electron density, or size of the secretory granule. In summary, our data show that 1) estradiol induces and maintains the mature epithelium of the baboon oviduct, 2) steroid withdrawal or the administration of progesterone causes regression of the epithelium, and 3) the previously identified estradiol-dependent oviduct-specific glycoproteins are synthesized within and released from the secretory epithelial cells.