The stages of the cycle in the rat seminiferous epithelium are illustrated for testes fixed by vascular perfusion and embedded in plastic resins. Improved cellular resolution in plastic sections permitted a clearer demarcation of the stages than in paraffin. Quantitative data are presented to support the recognition of stages, particularly those in transition. Stages IV, V, VII, XI, and XII had the highest frequencies of transitional characteristics. Stage IV was redefined to be more consistent with the occurrence of a high percentage of mitotic figures and to clarify transitions in this stage. Although the resolution of cellular detail was greatly improved with the use of plastics, the thinner sections contained fewer identifying features together within a single tubule cross section and sometimes major characteristics were absent. Therefore, additional characteristics were used for stage classification, such as nuclear diameter and the presence or absence of mitotic figures. A binary decision key is provided to improve consistency among laboratories in the identification of the stages in plastic-embedded testes.
A triple staining procedure was developed to evaluate bull spermatozoa using flow cytometry. Flow cytometric estimates of cell viability, measured by propidium iodide (PI) exclusion, and acrosomal integrity, measured by Pisum sativum agglutinin (PSA) binding acrosomal contents, were equivalent to estimates made by using standard laboratory assays. Mitochondrial function, measured by rhodamine 123 (R123) fluorescence, was depressed by the mitochondrial inhibitors rotenone (64%) or monensin (52%), establishing that mitochondrial damage can be detected. Dilauroylphosphatidylcholine (PC12) or lysophosphatidylcholine (LPC) was used to destabilize sperm membranes. When challenged with 15-30 microM PC12, selective exposure of PSA binding sites occurred without induction of PI uptake or loss of R123 staining. However, PC12 concentrations greater than 60 microM resulted in a loss of R123 fluorescence intensity. In contrast, greater than 1200 microM LPC was required to expose PSA binding sites, which also resulted in PI uptake. By using flow cytometry, these three stains in combination can be used to correlate three different features simultaneously on individual spermatozoa and assay thousands of cells per sample without extensive preparation.
Recent studies have implicated insulin-like growth factor I (IGF-I) as an intraovarian regulator of follicular growth and differentiation. Therefore, we investigated the possibility that cattle selected for twin births may have increased concentrations of IGF-I within the ovarian follicle and(or) in peripheral blood. The estrous cycles of 14 cows with histories of producing twins and 12 control monotocous cows were synchronized with 35 mg of prostaglandin F2 alpha (PGF2 alpha). Blood and follicular fluid were collected 48-50 h post-administration of PGF2 alpha (follicular phase of the estrous cycle). Concentrations of IGF-I were measured by RIA after acid-ethanol treatment of serum or follicular fluid. Twin-producing cows had a greater (p less than 0.05) number of large (greater than or equal to 4 mm) follicles and 47% greater (p less than 0.05) concentrations of IGF-I in peripheral blood than control cows. Cattle selected for high twinning frequency also had greater (p less than 0.05) concentrations of IGF-I (+/- SE) in the two largest follicles than control (unselected) cows (327 +/- 28 vs. 243 +/- 29 ng/ml). IGF-I concentrations in pooled small (1-3.9 mm) follicles were less (p less than 0.05) than in large follicles but did not differ between control and twin-producing cattle. In addition, the percentage of IGF-I concentrations measured in follicular fluid to that of serum was lower (p less than 0.05) in small follicles than in large follicles, and was greater (p less than 0.05) in large follicles of control (93.2 +/- 5.3%) than twin-producing (76.2 +/- 4.4%) cattle.
Human "spare" embryos, judged unsuitable for freezing because of their poor quality, were cocultured for 5 days on a "Vero" cell layer. These epithelial cells were selected because kidney and genital tract have a common embryologic origin and "Vero" cells are a safe and highly controlled cellular support used for vaccine production. In the control group, the embryos were cultured in culture medium alone (B2 + 15% serum). At the end of the culture, the number of blastocysts was significantly higher in the coculture group: 61% vs. 3%. Moreover, at least half of the blastocysts were expanding and hatching (13/25), with a chronologically normal development. These observations suggest that (1) the coculture system improves human embryonic development; (2) it can rescue early degenerating embryos; (3) beneficial effects of coculture are not strictly genital-tract specific, but rather epithelium dependent. This coculture system could be used for in vitro fertilization to prolong in vitro culture and thus make it possible to transfer embryos at a more appropriate time, to eliminate early-blocked eggs, and to freeze embryos at the blastocyst stage, when freezing procedures are most successful.
One-cell embryos from several different strains of mice have been cultured to the blastocyst stage in CZB medium. CZB medium can be used to culture CF1 x B6SJLF1/J 1-cell embryos to the blastocyst stage provided glucose is introduced into the medium on Day 3 of culture. The amount of glucose required for embryo development was titrated using a concentration range of 5.5 to 49.5 mM. With the exception of the highest concentration, all glucose levels tested supported 65-85% development to the morula and blastocyst stages. Variations of CZB medium were tested for their ability to support the development of 1-cell embryos from 4 strains of mice. For embryos from CF1 and DBA/2J (both x B6SJLF1/J) mice, which exhibit a "2-cell block" to development in vitro, CZB medium containing glutamine with the addition of glucose on Day 3 supported optimum development from the 1-cell stage to morula and blastocysts (79% and 87%). For embryos from B6D2F1/J and CD1 female mice (both x B6SJLF1/J males), which do not exhibit a "2-cell block" to in vitro development, optimum development to morula and blastocyst stages (95% and 50%) was in CZB medium containing both glutamine and glucose from the start of culture.
When hamsters mate shortly after the onset of estrus, spermatozoa are stored in the lower oviduct (isthmus) during the preovulatory period. The present study was performed to determine what proportion of the spermatozoa in the isthmus survive until fertilization. Females were mated 5 to 6.5 h before ovulation. When spermatozoa in the isthmus were observed through the wall of oviducts excised 2 h after the onset of mating, spermatozoa were seen free in the lumen, attached to the mucosal surface of the wall, and in crypts. The vast majority of spermatozoa in the lumen were immotile, whereas most of those attached to the mucosal surface of the wall and almost all of the those in the crypts exhibited flagellar movement. This suggested that attachment to the mucosa and/or storage in the crypts is beneficial to the survival of spermatozoa. Sequential flushing of an oviduct at various times (2-8 h) after mating was used to remove spermatozoa from the lumen (first flush), from the mucosal surface (second flush), and from the crypts (third flush). The highest number of spermatozoa was always contained in the first flush, the next highest in the second flush, and the smallest in the third flush. When Trypan blue was included in the flushing medium to differentiate live and dead spermatozoa, the first flush recovered the smallest percentage of liver spermatozoa (2-22%), the second flush slightly more (16-37%), and the third flush the highest (51-69%), regardless of the time after mating. These data indicate that the majority of spermatozoa stored in the hamster isthmus die before ovulation.
The trophoblastic interferons ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1, respectively) have been implicated as mediators of maternal recognition of pregnancy in sheep and cattle. The objective of this study was to describe the onset and duration of gene expression for oTP-1 and bTP-1 in preimplantation ovine and bovine conceptuses by in situ hybridization and Northern analysis. Sections from paraffin-embedded ovine conceptuses, collected on Days 10, 11, 12, 13, and 15 of gestation (n = 1, 3, 3, 2, 2), and bovine conceptuses, collected on Days 12/13, 15/16, and 19 (n = 2, 4, 5), were hybridized to specific [35S]-labeled cDNA probes. Two different probes, one encompassing bases 442-918 and representing both coding and 3'-untranslated regions, and a second 3'-specific probe (bases 650-912) were used to detect oTP-1 mRNA. At all stages examined, oTP-1 mRNA was confined to trophectoderm of ovine conceptuses. Consistent with earlier studies, expression increased markedly at Day 13. oTP-1 mRNA was detected at low levels in seven of seven ovine conceptuses prior to Day 13 when the longer probe was employed. With the 3'-specific probe, however, oTP-1 mRNA was detected in only one of the seven ovine conceptuses prior to Day 13. Thus, although low amounts of oTP-1 mRNA may be present in ovine conceptuses prior to Day 13, massive induction of this mRNA occurs on Day 13 coincident with the initiation of maternal recognition of pregnancy.
These studies were designed to develop a coculture system using a simple medium to promote development of 1-cell bovine embryos through the 8-16-cell stage to morula and blastocyst stages. Monolayers for coculture were prepared from bovine oviduct epithelial cells (BOEC). In vivo-fertilized 1-2-cell embryos and ova (384) were surgically collected from superovulated cows. In Experiment 1, embryos cocultured in a simple glucose-free and serum-free medium (CZB) developed with superior scores of embryo quality than embryos cocultured in Ham's F-10 with serum, and a greater percentage developed past 8-16 cells than embryos cocultured in CMRL-1066 with serum (p less than 0.05). In Experiment 2, embryos cocultured with fresh BOEC monolayers averaged more (p less than 0.05) cells than did embryos in coculture with frozen-thawed BOEC monolayers or in BOEC-conditioned medium. Without glucose in the simple medium for the first 48 h of culture, more embryos blastulated (p less than 0.01) by Day 5.5 of culture (Day 6.5 of donor's estrous cycle) than embryos in the same medium with glucose present throughout. In Experiment 3, more embryos tended to hatch in BOEC coculture (p less than 0.10) than in conditioned medium. These results show that a chemically simple medium with fresh BOEC monolayers can provide a significant benefit for coculture of early bovine embryos.
The Arg-Gly-Asp (RGD) sequence is known to play a role in many recognition systems involved in cell-to-cell and cell-to-matrix adhesion. In our experiments we demonstrated that an RGD-dependent recognition is involved in sperm-oolemmal adhesion and egg penetration. Following coincubation of RGD-containing oligopeptides in a heterologous system (human sperm and zona-free hamster eggs), a significant decrease in the number of oolemma-adherent sperm was noted at 15 microM RGDV (Arg-Gly-Asp-Val) and at 5 microM GRGDTP (Gly-Arg-Gly-Asp-Thr-Pro), and fertilization was completely inhibited at 250 microM RGDV and 30 microM GRGDTP. In a homologous system (hamster sperm and zona-free hamster eggs), a concentration-dependent decrease in oolemmal adhesion and egg penetration was also noted, with complete inhibition of fertilization at 200 microM GRGDTP. The specificity of the receptor was confirmed by the fact that small changes in aminoacid composition impaired the peptide's effectiveness and that peptide-dependent inhibition of fertilization was partially reversible in competition studies. The presence of a molecule on the oolemma capable of binding the RGD sequence was demonstrated by using immunobeads coupled with an RGD-containing hexapeptide (GRGDTP), which rosetted over the egg surface in a manner reversible by the addition of free GRGDTP in the medium.
Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozymewhich binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.
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.
The effect of vitamin A deficiency and vitamin A replacement on spermatogenesis was studied in mice. Breeding pairs of Cpb-N mice were given a vitamin A-deficient diet for at least 4 wk. The born male mice received the same diet and developed signs of vitamin A deficiency at the age of 14-16 wk. At that time, only Sertoli cells and A spermatogonia were present in the seminiferous epithelium. These spermatogonia were topographically arranged as single and paired cells and as clones of 4, 8 and more cells. A few mitoses of single, paired, and clones of 4 A spermatogonia were found, which were randomly distributed over the seminiferous epithelium. When vitamin A-deficient mice were treated with retinol-acetate combined with a normal vitamin A-containing diet, spermatogenesis restarted again synchronously. Only a few successive stages of the cycle of the seminiferous epithelium were present up to at least 43 days after vitamin A replacement. After 20 days, 98.3% of the seminiferous tubules were synchronized, showing pachytene spermatocytes as the most advanced cell type, mostly being in epithelium stages IX-XII. After 35 and 43 days, spermatogenesis was complete in 99.6% of the tubular cross sections, and most tubular cross sections were in stages IV-VII of the cycle of the seminiferous epithelium. The degree of synchronization was comparable or even higher than found in rats. The rate of development of the spermatogenic cells between 8 and 43 days after vitamin A replacement seemed to be similar to that in normal mice. Assuming that the rate of development of the spermatogenic cells is also normal during the first 8 days after vitamin A replacement, it can be deduced that the preleptotene spermatocytes, present after 8 days, were A spermatogonia in the beginning of stage VIII at the moment of vitamin A replacement. These results indicate that the mouse can be used as a model to study epithelial stage-dependent processes in the testis.
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.
The rat prostate is a complex ductal system with branches and subbranches extending from one end to another. Owing to the relative distance of various regions of the duct from the urethra, the entire length of the ductal system can be arbitrarily divided into three segments, i.e., the proximal, intermediate, and distal segments. The present study was carried out to assess the regional variation in cellular activities in this ductal system. Ventral prostates from adult Sprague-Dawley rats were dissected so that an individual ductal system was mechanically isolated and longitudinally sectioned to reveal various segments. Epithelial cells lining distal segments were tall-columnar type and were actively engaging in mitotic activity. Cells in intermediate segments were also tall-columnar type. However, they were mitotically quiescent, but able to produce secretory proteins. Evidence of programmed cell death was not observed in either of these two segments. Cells in proximal segments, on the other hand, were low-columnar or cuboidal in shape and were stained heavily for cathepsin D, a marker associated with late manifestation of cell death. Following castration in adult rats, there was a reversal in the site of programmed death in cells lining the ductal system. By Day 4 post-castration, distal segments contained many epithelial cells with intense cytoplasmic staining for cathepsin D while proximal segments showed a reduction in number of positively stained cells. By Day 7 post-castration, cells in proximal segments, though atrophied, were devoid of staining for cathepsin D.
Procedures to improve nuclear transplantation efficiency in the rabbit were evaluated. We report the influence of recipient oocyte age on the different steps of nuclear transplantation. The effect of multiple pulses and the influence of manipulation medium and cytochalasin B in the post-fusion/activation medium on activation and development were studied. Recently ovulated oocytes were enucleated at a higher rate (60%) than aged oocytes (3%, p less than 0.005); they also fused at a higher rate (85% vs. 26%, p less than 0.001). Activation was low with freshly ovulated oocytes compared to aged oocytes (3% vs. 37%, respectively; p less than 0.005), but was increased by using multiple pulses (85% vs. 68%, p less than 0.05). Multiple pulses also improved development to blastocysts (48% vs. 5%, p less than 0.001). Incubation of oocytes in a bicarbonate-buffered medium with 10% fetal calf serum for manipulation also enhanced rates of activation (100% vs. 89%, p less than 0.05) and development of oocytes to blastocysts (77% vs. 26%, p less than 0.001). Furthermore, 7.5 micrograms/ml cytochalasin B in the post-fusion/activation medium increased activation rates (78% vs. 50%, p less than 0.05) and development to blastocysts of manipulated embryos (46% vs. 11%, p less than 0.001). When the above modifications were applied, 10% (23/230) of the total nuclear transplant embryos (8-16-cell-stage donor nuclei) or 21% (23/110) of those transferred to recipients developed to offspring, rates similar to the development of nonmanipulated control embryos (10%, 4/41, p greater than 0.1).
The potential involvement of macrophages, T lymphocytes, and the cytokine tumor necrosis factor (TNF) in regression of the corpus luteum was investigated at different stages of pseudopregnancy and pregnancy by use of immunocytochemical methods and a TNF bioassay. Few macrophages (11 +/- 6 per high power field of 8-microns frozen sections of corpus luteum, Day 10 of pseudopregnancy) were observed until the very end of pseudopregnancy, when the number of macrophages increased greatly (176 +/- 42 per high power field, Day 19 of pseudopregnancy). Pregnancy, of 32 days duration, delayed large-scale macrophage accumulation until 3 days after parturition (154 +/- 30 per high power field). Low TNF activity (approximately 1.0 U/mg protein) was detected in incubations of luteal tissue at all stages; in response to lipopolysaccharide, TNF values in medium increased 10- to 30-fold at times of luteal regression and macrophage accumulation (1 day postpartum and Day 19 of pseudopregnancy). Class II-positive T lymphocytes were observed in luteal tissue, but unlike macrophages, the number of lymphocytes did not increase at the time of regression of the corpus luteum. These data are consistent with the hypothesis that involution of the corpus luteum is promoted through the interactions of inflammatory cells and action of TNF, although the action of TNF has not been determined in this luteal tissue. Through unknown mechanisms, pregnancy postpones the accumulation of macrophages in the corpus luteum, in association with the prolongation of luteal function until the time of parturition.
The biogenesis of the sperm-specific organelle, the acrosome, was investigated using an acrosomal glycoprotein as a marker of development. This component, which we have named acrogranin, was purified from an acid extract of guinea pig testes by standard chromatographic procedures. The molecular weight of reduced acrogranin was determined to be 67,000 by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunization of female rabbits with purified acrogranin produced an antiserum that recognized a single protein with Mr = 67,000 in an acid extract of guinea pig testes. By indirect immunofluorescence, acrogranin was found only in the acrosome of mature sperm. In haploid spermatids, acrogranin was localized in the developing acrosome and, weakly, in the cytoplasm. Acrogranin was also detected in the cytoplasm and juxtanuclear region in putative proacrosomal granules of meiotic cells (pachytene spermatocytes). Detergent extracts from different purified germ cell populations contained only the Mr = 67,000 form of acrogranin, but sperm extracts had four lower Mr immunoreactive forms not present in the testicular extracts. By two-dimensional gel electrophoresis, acrogranin was found to be an acidic glycoprotein. Analysis of glycosylated and trifluoromethanesulfonic acid-deglycosylated acrogranin indicated that the antibody recognized polypeptide determinants. After highly enriched germ cell populations were labeled overnight with [35S]methionine and extracted with detergent, anti-acrogranin immunoprecipitated a single protein of Mr = 67,000. The synthesis of acrogranin by pachytene spermatocytes and round spermatids was similar, but the synthesis of the glycoprotein by condensing spermatids was markedly reduced. These studies demonstrate that acrosome biogenesis, as determined by the synthesis of a specific acrosomal component, begins during meiosis and continues through the early stages of spermiogenesis.