Cross-talk between free and bound spermatozoa to modulate initial sperm:egg ratios at the site of fertilization in the mammalian oviduct

This essay proposes that highly localized communication between free and bound spermatozoa in the caudal portion of the oviduct acts to regulate the numbers detaching from the epithelium and progressing to the site of fertilization close to the time of ovulation. Low initial sperm:egg ratios are essential for monospermic fertilization. Liberation of surface macromolecules and metabolic prompting from activated spermatozoa, together with altered patterns of sperm movement and dynamic differences in intracellular Ca²⁺ ion status between neighboring sperm cells, would influence the progressive release of spermatozoa from the reservoir in the oviduct isthmus. Different intensities of preovulatory epithelial binding, reflecting a range of states in the sperm surface membranes and associated proteins, would provide a further explanation for a chronologically staggered periovulatory detachment of spermatozoa. Intimate sperm–sperm interactions within the confines of the oviduct isthmus offer a sensitive means of fine-tuning the vanguard of competent male gametes reaching the isthmo-ampullary junction.     

Fertilizing capacity of bovine sperm may be maintained by binding of oviductal epithelial cells

The ability of the bovine oviduct to maintain the motility and fertilizing capacity of bovine sperm was investigated by incubating frozen-thawed sperm with endosalpingeal epithelial cells cultured on either tissue culture plastic (nonpolarizing) or Matrigel-coated Millicell (polarizing) substrata. Sperm were also incubated in medium alone or with cultured bovine tracheal epithelial cells. Motility was determined at 6-h intervals over a 48-h period. The fertilizing capacity of sperm was evaluated after 0, 24, and 30 h of incubation by adding oocytes to the culture and determining the incidences of fertilization and polyspermy. Motility was maintained for 48 h in sperm that bound to endosalpingeal epithelial cells, but to a greater extent with polarized cells (38.4% motile) than with nonpolarized cells (0.8%). Fertilizing capacity was maintained for 30 h in sperm incubated with endosalpingeal epithelial cells on Matrigel/Millicell, but not in sperm incubated in medium alone or with tracheal cells. Only sperm incubated with oviductal cells developed hyperactivated motility. Scanning electron micrographs revealed that sperm were bound by the rostral portion of the intact acrosome to the apical surface of polarized endosalpingeal cells. These results suggest that the oviduct may not only store sperm but may also maintain sperm viability and fertilizing capacity during the preovulatory period.     

Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS-2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell-conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO₂ in air. Viability and the occurrence of true acrosome reactions were assessed using a triple-stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS-2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS-2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS-2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley-Liss, Inc.     

In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, protein-free culture media.

Development of bovine embryos derived from in vitro-matured/in vitro-fertilized (IVM/IVF) oocytes was examined in two culture media: hamster embryo culture medium (HECM), a relatively simple, chemically defined, protein-free medium containing 20 amino acids; and tissue culture medium (TCM)-199, a more complex medium designed for culture of somatic cells. The first experiment studied (1) effects of glucose and/or phosphate (Pi) using HECM and (2) the development of bovine IVM/IVF embryos in four different conditions: HECM, TCM-199, TCM-199 + 10% unheated bovine calf serum (BCS), and oviduct cell-conditioned TCM-199 + 10% BCS. After IVF, 45% of the inseminated oocytes developed to the morula/blastocyst stages (M&B) when cultured in HECM; blastocyst development was depressed in the presence of glucose and Pi when compared to Pi alone. When the four culture conditions were compared, there was no significant difference in M&B development (42-51% of inseminated oocytes). However, blastocyst development in TCM-199 supplemented with 10% BCS (29.7%) or with BCS + oviduct cell-conditioned medium (21.6%) was significantly greater than in nonsupplemented HECM (9.7%) or TCM-199 (13.8%). In the second experiment, the effects of serum supplementation and/or oviduct cell conditioning on HECM and TCM-199 were examined in a 2 x 2 x 2 factorial experiment. Oviduct cell conditioning of either HECM or TCM-199 without serum supplementation did not enhance bovine embryo development. Serum supplementation exhibited a biphasic effect, with inhibition at the first cleavage and stimulation of morula compaction and blastocyst formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sperm binding to epithelial oviduct explants in bulls with different nonreturn rates investigated with a new in vitro model

A new in vitro method was developed for analyzing the capacity of sperm to bind to oviductal epithelium to determine whether this binding capacity could be used to predict nonreturn rates (NRR). Sperm binding was evaluated by counting 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)-labeled spermatozoa attached to oviductal epithelium and by measuring the surface area of the oviduct explants by means of an image analysis program. Hepes + Tyrode albumin lactate pyruvate (TALP) was a more useful medium than in vitro fertilization (IVF)-TALP, TCM-199 medium + 10% fetal calf serum, and TCM-199 medium alone for the investigation of sperm binding to oviductal explants. Oviduct explants with a surface area of < 20 000 micro m(2) provided more consistent results than did explants with a surface area of >100 000 micro m(2). A positive association was found between the log(e) transformed number of spermatozoa bound to 0.1 mm(2) oviductal epithelium and the NRR of the respective sires after 24 h of coincubation, provided that the membrane integrity of the sperm sample was >60%. Determination of the capacity of sperm to bind to oviductal explants could become a reliable in vitro method for predicting the NRR of a given sire.     

Development of bovine embryos after in vitro fertilization of oocytes with flow cytometrically sorted, stained and unsorted sperm from different bulls

The objective of this study was to determine the effect of staining bovine sperm, with or without flow cytometry, on in vitro fertilization of bovine oocytes and blastocyst development. Bovine oocytes (n=4273) were fertilized with frozen–thawed sperm from three bulls that was: stained with Hoechst 33342 and sorted (into X- or Y-chromosome-bearing sperm) with flow cytometry; stained but not sorted; and not stained or sorted (Control). Oocytes, aspirated from slaughterhouse ovaries, were matured in TCM199 (supplemented with 10% fetal calf serum and 15 ng FSH, 1.0 μg LH, 1.0 μg E2/ml) for 22–24 h at 39 °C in 5% CO₂ in air with maximum humidity. Presumptive zygotes were removed from culture and placed in chemically defined medium (CDM-1) 6–7 h after insemination and cultured for 65–66 h. Embryos that had cleaved by 72 h post-insemination were cultured an additional 96 h in CDM-2 containing 0.12 IU insulin/ml. Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7–8 after insemination, respectively. There was no significant difference in blastocyst rate among the three types of sperm; however, cleavage rates with stained and sorted sperm (53.1%) and unsorted, stained sperm (59.9%) were lower (P<0.05) than Control sperm (69.7%). Furthermore, there were significant differences due to semen from different bulls in cleavage and blastocyst rates.     

Bovine oviduct-specific glycoprotein: A potent factor for maintenance of viability and motility of bovine spermatozoa in vitro

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. © 1995 wiley-Liss, Inc.     

In vitro culture of sheep oocytes matured and fertilized in vitro

Sheep oocytes that matured and fertilized in vitro were cultured to evaluate their cleavage to the 8- to 16- cell stage and further development in five different media as follows: 1) CPMW (TCM199 + 20% ewe serum + 0.4% BSA), 2) Ham's F-10 + 10% ewe serum, 3) Brinster's pyruvate medium + 0.1% glucose (BPM-G), 4) co-culture with sheep oviduct epithelial cells in TCM199 + 10% fetal calf serum, and 5) co-culture with sheep granulosa cells in the same medium as 4. The culture duration was 4 or 7 d for 8- to 16-cell or further development. The proportions of 8- to 16-cell eggs were 1) 16% (8 49 ), 2) 25% (12 49 ), 3) 52% (58 112 ), 4) 63% (105 167 ) and 5) 45% (27 60 ). The co-culture with sheep oviduct cells resulted in a significantly (P < 0.05) higher rate of cleavage than the other media, except BPM-G. The proportion of noncompacted morula (35%, 24 68 ) was also significantly (P < 0.05) higher in the co-culture of sheep oviduct cells than the other media. The 8- to 16-cell eggs produced by BPM-G (n=38) and the co-culture with sheep oviduct cells (n=42) were transferred into the uterus of recipient ewes, but no elongated blastocysts were obtained 13 d later. On the other hand, 8 out of 55 one-cell eggs (15 to 18 h after in vitro insemination) transferred to the oviduct of recipient ewes were elongated blastocysts (24% of 34 recovered eggs). The data show that the co-culture of in vitro fertilized eggs with sheep oviduct epithelial cells could support development of 8- to 16-cell embryos or early morula, but their viability is still questionable.     

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.     

In vitro development of bovine embryos in Buffalo rat liver- or bovine oviduct-conditioned medium

A culture system for bovine embryos was developed using Buffalo rat liver cell (BRL) line-conditioned medium without serum. Zygotes, obtained by in vitro maturation and fertilization of oocytes, were cultured either in unconditioned medium (TCM 199 or DMEM/F12) or in the same medium conditioned by bovine oviduct or BRL cells. No serum was added during conditioning or during embryo culture. The DMEM/F12 medium was superior to TCM 199 for development of bovine embryos to the 5 to 8-cell stage: on average between 50 and 57% of the embryos reached this stage after 2 d of culture in DMEM/F12 or in conditioned medium, while 36% reached this stage in TCM 199. Further development to the blastocyst stage was enhanced by conditioning. The highest percentage of blastocysts was achieved in DMEM/F12 medium conditioned with BRL cells (30%). The yield of blastocysts was similar in TCM 199 and in DMEM/F12 media conditioned with bovine oviduct cells (22 versus 20%), but after conditioning with BRL cells, DMEM/F12 medium yielded a higher percentage of blastocysts than TCM 199 (30 versus 18%). This might be explained by the fact that viability of BRL cells was better in DMEM/F12 medium than in TCM 199 when serum was omitted. Blastocysts produced in BRL-conditioned medium had a higher number of cells than blastocysts obtained in bovine oviduct-conditioned medium, and their transfer to recipients led to pregnancies and birth of calves. In conclusion, culture of bovine embryos in DMEM/F12 medium conditioned with BRL cells without serum led to the development of good-quality blastocysts and is thus a promising method for producing embryos for the study of potential embryotrophic factors. The use of rat liver cell lines guarantees against bovine viruses and allows for better production of embryos.     

Molecules involved in sperm-oviduct adhesion and release

In mammals, sperm ascension within the female reproductive tract involves a transient adhesion to the caudal isthmus of the oviduct. Sperm adhesion to this specialized region, which is termed the “oviductal reservoir”, extends the sperm fertile life span by delaying capacitation until, around ovulation, specific signals induce sperm release. In vivo and in vitro studies demonstrated that carbohydrates on the oviductal cell apical membranes and lectin-like molecules on the rostral sperm surface are involved in adhesion in a species-specific way. In this respect, the most intensely studied species are pigs and cattle. On the other hand, less is known about molecules involved in sperm release. Direct evidence that molecules present in the oviductal fluid trigger the release of sperm bound to in vitro cultured oviductal epithelium has been provided only in cattle. However, the identity of sperm and/or oviductal molecules that respond to these releasing signals is still unknown. The comprehension of molecular mechanisms underlying sperm-oviduct interaction may advance our understanding of the behavior of sperm within the female reproductive tract and provide new tools for sperm selection, extension of fertile life and modulation of capacitation in the field of reproductive biotechnologies. The aim of the present paper is to review the available knowledge on molecules involved in sperm selection, storage and release from the oviductal reservoir.     

Ventricular and atrial myocytes of newborn rats synthesise and secrete atrial natriuretic peptide in culture: Light-and electron-microscopical localisation and chromatographic examination of stored and secreted molecular forms

Summary We have demonstrated that atrial natriuretic peptide-like immunoreactivity is stored and secreted by ventricular and atrial myocytes in dissociated cell culture preparations from the heart of newborn rat. Culture preparations were maintained in either foetal calf serum-supplemented medium 199 or in hormone-supplemented, serum-free medium 199. The presence of atrial natriuretic peptidelike immunoreactivity in the cultured myocytes was demonstrated at both light-and electron-microscopical levels. Release of atrial natriuretic peptide-like immunoreactivity into the culture medium was measured by radioimmunoassay; molecular forms of the stored and secreted peptide were determined by gel column chromatography. The atrial natriuretic peptide-like immunoreactivity of cultured atrial and ventricular myocytes was concentrated in the perinuclear cytoplasm and was localised to electron-dense secretory granules. The number of immunoreactive ventricular myocytes and the intensity of their immunofluorescence changed with time in culture and was higher in cultures in foetal calf serum-supplemented medium than in serum-free medium. Gamma-atrial natriuretic peptide was stored and released by cultured atrial and ventricular myocytes, but was broken down to alpha-atrial natriuretic peptide in the growth medium. This process was foetal calf serum-independent, since it occurred in both the media used, indicating that cardiac myocytes in culture may release a factor that cleaves gamma-atrial natriuretic peptide to form alphaatrial natriuretic peptide.     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 3. In vitro fertilisation using sheep serum as a capacitating agent and the subsequent birth of calves

The following experiments investigated the use of sheep serum (SS) as a capacitating agent for red deer (Cervus elaphus) sperm during in vitro fertilisation. Red deer oocytes were collected at slaughter and matured in vitro for 24h in TCM-199 supplemented with 10% foetal calf serum, 10 microg ml(-1) FSH and LH, and 1microg ml(-1) of oestradiol. Fertilisation medium was IVF-SOF modified to contain 5mM Ca(2+) and no glucose. Experiment 1 investigated the addition of heparin, BSA (8 mg ml(-1)) or 20% SS. All oocytes were penetrated when IVF-SOF was supplemented with SS compared to 10 and 0% penetration when either heparin or BSA was present (P<0.01). However, 43.8% of these oocytes were polyspermic when the medium contained SS. In Experiment 2, the effect of sperm concentration on penetration rates during in vitro fertilisation was investigated. Total sperm penetration and monospermic penetration rates increased with increased sperm concentrations in a log linear manner (P<0.001) and both approached an asymptote at 0.4 x 10(6) sperm ml(-1) with 93.6 and 77% for total and monospermic penetration, respectively. Polyspermic fertilisation also increased with increasing sperm concentrations (P<0.05) but was variable (range 3.5+/-4.2 to 42.3+/-10.6%), especially at the lower sperm concentrations. Experiment 3 investigated the viability of these oocytes after transfer into red deer recipients. Fifteen 2- and 4-cell embryos were transferred into the oviducts of synchronized recipients 28 h post in vitro insemination. An additional fourteen embryos (8-10 cell) were transferred into synchronised recipients after 48 h of in vitro culture in either SOFaaBSA (n=10) or on red deer epithelial oviduct monolayers (n=4). Five (33% 5/15) of the recipients that received 2- and 4-cell embryos were pregnant at Day 45 (verified by ultrasonography) and four recipients subsequently calved. One recipient receiving an embryo cultured in SOFaaBSA was pregnant at Day 45 and subsequently calved. The birth of five normal calves indicate that full developmental competence of red deer oocytes matured and fertilised in vitro can be achieved by the techniques described.     

Effect of bovine oviduct epithelial cell apical plasma membranes on sperm function assessed by a novel flow cytometric approach

In the bovine, as in many mammalian species, sperm are temporarily stored in the oviduct before fertilization by binding to the oviduct epithelial cell apical plasma membranes. As the oviduct is able to maintain motility and viability of sperm and modulate capacitation, we propose that proteins present on the apical plasma membrane of oviduct epithelial cells contribute to these effects. To verify this hypothesis, the motility of frozen-thawed sperm was determined after incubation for 6 h with purified apical plasma membranes from fresh or cultured oviduct epithelial cells or from bovine mammary gland cells as a control. Analysis of intracellular calcium levels was performed by flow cytometry on sperm incubated with fresh membranes using Indo-1 to assess the membrane effect on intracellular calcium concentration. The coculture of sperm with fresh and cultured apical membranes maintained initial motility for 6 h (65% and 84%, respectively). This effect was significantly different from control sperm incubated without oviduct epithelial cell apical membranes (23%), with mammary gland cell apical membranes (23%), or with boiled epithelial cell apical membranes (21%). Apical membranes from oviduct epithelial cells diminished the percentage of sperm that reached a lethal calcium concentration over a 4-h period (18.7%) compared with the control (53.8%) and maintained lower intracellular calcium levels in viable sperm. These results show that the apical plasma membrane of bovine oviduct epithelial cells contains anchored proteinic factors that contribute to maintaining motility and viability and possibly to modulating capacitation of bovine sperm.     

Influence of female age, sperm senescence and multiple mating on sperm viability in female Drosophila melanogaster

Sperm viability has been associated with the degree of promiscuity across species, as well as the degree of reproductive success within species. Thus, sperm survival within the female reproductive tract likely plays a key role in how mating systems evolve. In the fruit fly, Drosophila melanogaster, however, the extent and cause of sperm death has been the subject of recent debate. Here, we assess sperm death within the female reproductive tract of D. melanogaster following single and multiple matings in order to elucidate the extent of death and its potential mechanisms, including an acute female response to mating, female age and/or sperm senescence. We found no evidence that sperm viability was influenced by an acute female response or female age. We also found that rival ejaculates did not influence viability, supporting recent work in the system. Instead, the majority of death appears to be due to the aging of male gametes within the female, and that at least some dead resident sperm remain in the female after multiple mating. In contrast to earlier in vivo work, we found that overall sperm death was minimal (8.7%), indicating viability should have a negligible influence on female remating rates.     

An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture

The gastric surface epithelium is situated at an air-liquid interface because the luminal surface of the alimentary tract is in continuity with the air phase. However, the effects of this microenvironment on the gastric epithelium remain unclear. The aim of this study was to clarify the effects of an air-liquid interface on gastric epithelial cell biology. Gastric surface mucous cells (GSM06) were cultured at an air-liquid interface. Cultured cells were examined by histology, histochemistry, and transmission electron microscopy. When the cells were cultured at an air-liquid interface, the surface cells on the collagen gel became tall columnar and secreted periodic acid-Shiff-positive substances at the apical surface. These cells indicated many mucous granules in the apical cytoplasm and organized the basal lamina at the contact side with the gel. In contrast, under immersed condition, the surface cells showed immature features. This is the first report of an air-liquid interface promoting the differentiation of gastric surface mucous cells in a reconstruction culture of the gastric surface epithelial layer, suggesting that an air-liquid interface may function as a crucial luminal factor to maintain the homeostasis of gastric mucosa.     

Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells

One-cell bovine embryos fertilized in vivo were cultured in TCM-199 and bovine oviductal epithelial cells, in TCM-199, or in explanted immature mouse oviducts supported by TCM-199 to compare development to the blastocyst stage. The morphological stage of development and cell number were determined following 144 hours of culture. Of the embryos that cleaved at least once, 52.6, 30.4 and 0.0% developed to the morula/blastocyst stage after culture in oviductal epithelial cells, in TCM-199 alone, or in explanted mouse oviducts, respectively. The mean total cell number for embryos cultured in oviductal epithelial cells (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<0.05). The mean cell number of embryos cultured in TCM-199 or in explanted mouse oviducts did not differ. The explanted immature mouse oviduct supported by TCM-199 did not provide an environment adequate for development of one-cell bovine embryos to the blastocyst stage. Development of one-cell bovine embryos was best supported by co-culture with oviductal epithelial cells in TCM-199 medium.     

Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells

To examine the effects of somatic cell support on the cleavage and viability of fertilized sheep eggs, 434 pronucleate eggs were co-cultured for 3 or 6 days on oviduct cells or fibroblasts and 77 eggs were cultured in medium alone. During the first 3 days in culture 95% of the single-celled eggs cleaved regularly to non-compacted morulae on either of the feeder-layers but only 13% underwent similar regular cleavage in medium alone. Despite the identical cleavage rates in the co-culture groups, only 33% of embryos grown on fibroblasts as compared with 80% of embryos grown on oviduct cells were fully viable as judged by their ability to develop normally after transfer to recipient animals. The viability of embryos in the oviduct group was equal to that obtained after the direct transfer of morulae from donor to recipient sheep. After 6 days in culture 42% of embryos co-cultured with oviduct cells developed into expanded blastocysts as compared with only 4.5% cultured on fibroblasts. In both co-culture groups virtually all the remaining embryos blocked during the 4th cleavage. When transferred, 30% of blastocysts grown from the pronucleate stage on oviduct cells were viable. We conclude that: (1) during the first 3 days after fertilization cleavage will progress at a normal rate on different feeder-layers but oviduct cells appear to be required for the acquisition of full embryonic viability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human epidermis reconstructed on synthetic membrane: Influence of experimental conditions on terminal differentiation

Summary Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham’s F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham’s F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to thestratum spinosum, stratum granulosum, andstratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no properstratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham’s F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space. Conversely, differentiation appears to depend on elements of basal medium and/or components synthesized by keratinocytes under the influence of the culture medium.     

Maturation and sperm penetration of canine ovarian oocytes in vitro.

Canine ovarian oocytes were cultured in a medium consisting of TC medium 199, fetal calf serum and antibiotics. Ninety-nine percent of the apparently healthy oocytes were in the germinal vesicle (dictyate) stage when recovered from the ovaries; 25% of them reached metaphase I or II by 72 hours of culture. Washed ejaculated spermatozoa were added to BWW medium containing oocytes which had either been removed directly from the follicles or which had been cultured for 24--72 hours. The earliest acrosome reaction and zona penetration by spermatozoa were seen at seven hours after insemination. Seventy-four percent of the oocytes examined between 11 and 24 hours after insemination showed evidence of zona penetration by spermatozoa. Neither the condition of the oocyte vitellus nor the stage of nuclear maturation influenced the incidence of zona penetration. Decondensing sperm nuclei were found in the vitellus of 27% of the oocytes which had not been cultured and in the vitellus of 20% of those which had been cultured for 24--72 hours and were in various stages of maturation. These results indicate that (1) canine ovarian oocytes can be matured in vitro, (2) the spermatozoa require capacitation which takes approximately seven hours in vitro and (3) maturation of the oocytes is not required for sperm passage through the zona pellucida or entry into the vitellus nor for sperm nuclear decondensation.