Duration of premeiotic deoxyribonucleic acid synthesis and the stages of prophase I in rabbit oocytes.

To estimate the duration of oocyte DNA synthesis 36, 3-day-old female rabbits received 3, 6, 9, 12, 15, or 18 injections of tritiated thymidine (thy-(3)H) at hourly intervals. The ovaries, removed at 1, 10, or 20 days after the first injection, were radioautographed. Counts made of the number of silver grains associated with oocyte nuclei in meiotic Prophase I indicate that the duration of DNA synthesis is between 9 and 12 hr. To determine the length of the stages of meiotic Prophase I, a group of 2-3-day-old rabbits was given a single sub-cutaneous injection of thy-(3)H, and the ovaries were removed at hourly and/or daily intervals after treatment. The minimum duration of leptotene was 3 hr and the maximum duration probably was less than 8 hr. The maximum durations of zygotene, pachytene, and diplotene were estimated to be 44, 216, and 96 hr, respectively. The interval from the end of oogonial DNA synthesis to the beginning ofpremeiotic DNA synthesis (G(2) + Mitosis + G(1)) appeared to be less than 6 hr.

Age-associated changes in nuclear binding of rat uterine estradiol receptor complexes.

Nuclear binding of cytoplasmic estrogen receptors was measured in an in vitro cell-free system, using various mixtures of cytosols and nuclei from uteri of mature (6-9 month old) and senescent (24-25 month old) Wistar rats. Both nuclei and cytoplasmic receptors from senescent uteri were 25-35% less efficient in supporting nuclear binding than those obtained from mature tissues as evidenced by the concentrations of occupiable nuclear acceptor sites. No age differences in association or dissociation constants were observed for the nuclear binding reaction. However, the apparent inability of some aged receptors to bind to the full complement of mature nuclear acceptor sites may indicate a qualitative deficiency in the cytosols of senescent uteri.

Effects of in vivo estradiol administration on availability of rat uterine nuclear acceptor in measured in vitro.

Estradiol injected in vivo successfully completes for binding to uterine nuclear acceptor sites measured by the assay of Kon and Spelsberg (1). Such competition is time-and dose-dependent, with maximal inhibition (75%) 1 h after injection and at a dose of 10 micrograms of 17 beta-estradiol per 300 g of BW. Thus, this assay appears to accurately measure those uterine nuclear acceptor sites mediating estrogen action in vivo.

Factors limiting reproductive efficiency in selected laboratory animals.

The mouse, rat, hamster, and rabbit have had remarkable success in surviving in the wild, which attests to their high reproductive capability. In fact, Parkes refers to mice conceiving at each postpartum estrus having the potential for 13 litters per year. Paufler et al. used GnRH to repeatedly cause ovulation in 27 rabbits a few days after parturition, resulting in an average pregnancy rate of 71.5%, 7.0 young born, 5.8 young weaned, and 50 young per doe per year. All four species produce an excess of spermatozoa relative to the requirements for fertilization. The rabbit is suitable for semen collection, artificial insemination, and most of the techniques one might wish to model for reproductive studies in both males and females. A major limitation to reproduction in these species is that reproductive capacity fails long before the finite oocyte population formed prenatally is depleted. The uterus of the aged female appears to be the major cause of reproductive failure, as fertilized eggs replaced in such a uterus usually soon deteriorate. With in vitro techniques, much is still to be learned about harvesting, maturing and fertilizing oocytes and identifying those most likely to result in formation of a healthy neonate.

Bovine oviductal epithelial cells (BOEC) and oviducts: I. For embryo culture. II. Using SEM for studying interactions with spermatozoa.

The oviduct (uterine tube) plays a major role in reproduction. It is a dynamic organ which selectively permits a few sperm to undergo capacitation and reach the oocyte which has continued to undergo maturation following ovulation. Then following fertilization the embryo undergoes cleavage before arriving in the uterus. Extensive information has become available from in vitro studies on oocytes as well as spermatozoal interactions with oviductal cells. Bovine oviduct epithelial cell (BOEC) monolayers with simple media provide an environment in which zygotes can be cultured to blastocysts in 6 days with cell numbers essentially equivalent to blastocysts grown totally in the donor animal. These yield normal pregnancy rates upon transfer. The simple protein-free media currently under test hold promise for elucidating specific requirements of the preimplantation embryo and these defined conditions facilitate many related studies on in vitro fertilization and genetic engineering of embryos. The second part of this paper is an extensive study on the interaction of fresh and frozen-thawed bull spermatozoa with BOEC and segments of intact oviducts as viewed by SEM. Both types of oviductal cells were incubated at 39 degrees C for 0, 3, 6, and 9 hours, using material obtained from periovulatory cows. Sperm attached immediately to both types of epithelium and reached a peak at 3 hours. They were found primarily in the furrows of the intact oviducts. Secretory droplets appeared rapidly on the anterior portion of the sperm head and acrosomal changes were evident in 3 hours, similar to those reported in vivo. Changes were more rapid with frozen-thawed sperm.

A procedure for cryopreservation of hamster oocytes yielding highly conserved oocytes suitable for sperm penetration tests.

Hamsters were superovulated and their oocytes frozen in Dulbecco's phosphate-buffered saline containing 10% fetal calf serum and 1.5 molar 1,2-propanediol. In several experiments, 94% of the hamster oocytes survived freezing and were equivalent to fresh oocytes in sperm penetration. In routine use of the procedure described, also 94% of 1,340 frozen-thawed oocytes were satisfactory for sperm penetration of several species. Frozen oocytes eliminate problems and costs of maintaining hamster colonies with variable responses on individual days.

Repeatability of measurements on human, rabbit, and bull sperm by computer-assisted sperm analysis when comparing individual fields and means of 12 fields.

OBJECTIVE: To compare repeatability of measurements of human, rabbit, and bull sperm on two Hamilton Thorne Integrated Visual Optical System (IVOS) units, software version 10. DESIGN: Semen samples from seven normal human subjects, six rabbits, and eight bulls were obtained at regular intervals. The samples were diluted, two chambers were filled, and 12 fields were recorded, using high resolution recorders. Computer-assisted sperm analysis (CASA) was performed nearly simultaneously with two Hamilton Thorne IVOS units. SETTING: Reproduction Research Laboratories, Cornell University and Hamilton-Thorne Research, Beverly, Massachusetts. RESULTS: Optimal settings were established for evaluating by CASA sperm from three species. Fifteen variables were analyzed. The correlation coefficients for most variables characterizing sperm motion and concentration, when means of 12 fields were calculated, were 0.95 to 1.00. There were too few hyperactive sperm to obtain a reliable correlation for human sperm (r = 0.63) and repeatability of elongation was lower only for human sperm (r = 0.75). CONCLUSIONS: Two units of Hamilton Thorne IVOS, software version 10, were capable of providing nearly identical estimates of many CASA variables of human, rabbit, and bull sperm. Correlations for the paired estimates of many motion characteristics and sperm concentration usually exceeded 0.97.

Motility and other characteristics of human sperm can be measured by computer-assisted sperm analysis of samples stained with Hoechst 33342.

OBJECTIVE: To develop methods for using a DNA-specific dye to discriminate between motile and nonmotile sperm and static particulate matter in fresh and diluted semen, using computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, TOX version (Hamilton-Thorne Research, Beverly, MA). DESIGN: Donor semen was divided for treatment as fixed stained sperm (Hoechst 33342 stain; Sigma Chemical Company, St. Louis, MO), fresh motile and nonmotile stained sperm, and unstained control sperm. SETTING: Normal human volunteers in an academic research and medical environment. PATIENTS: Selected healthy student volunteers. INTERVENTIONS: Delivered semen to the laboratory within 1 hour of collection. MAIN OUTCOME MEASURE: Semen quality measured by CASA. RESULTS: Fixed or fresh human sperm stained with Hoechst 33342 dye should be diluted to < or = 50 x 10(6) sperm/mL to count sperm accurately. Motile and nonmotile sperm were stained suitably with 5 to 10 micrograms/mL of dye when diluted with a simple diluent, but the dye concentration should be increased to 40 micrograms/mL when egg yolk is in the diluent. CONCLUSIONS: The DNA-specific dye, Hoechst 33342, can be used to discriminate between motile and nonmotile sperm and other particulate matter when evaluated by CASA with instrumentation equipped with suitable optics.

Embryo mortality and altered uterine luminal proteins in progesterone-treated rabbits.

This study was undertaken to elucidate the location, time, and nature of embryo mortality induced by preovulatory progesteron administration. Progesterone was injected into rabbits on days -2, -1, and 0 (the day of mating) at doses of 0.5, 1.0, and 1.0 mg, respectively. Normal fertilization rates resulted, but embryonic death occurred by day 4. Embryos residing in progesterone-treated does for up to 3 days survived normally when transferred to normal recipients, whereas day 4 embryos from treated does exhibited a reduced ability to implant. Uterine fluid (UF) protein pattern was examined on days 1 to 7 after mating. Total UF protein levels were significantly greater in treated animals on day 4 than in controls. Uteroglobin secretion was significantly advanced in the treated animals by day 3. Examination of the time of the arrival of embryos into the uterus revealed a delay in the progesterone-treated rabbits. This delay, coupled with the earlier secretion of uteroglobin in the treated rabbits, indicated a possible asynchrony of approximately 1 day between embryo arrival in the uterus and certain uterine proteins. To determine whether UF could be etiologically implicated in the progesterone-induced embryonic death, embryonic development in vitro and in vivo was examined after exposure to UF collected at different gestational stages. More normal day 3 morulae placed in UF from day 3 control and day 2 progesterone-treated rabbits developed than similar morulae placed in UF from day 2 controls and day 3 progesterone-treated does. Hence, partial physiologic synchrony was achieved. This was interpreted to mean that "asynchronous" UF can be embryotoxic. Infertility was transient. Ability of the does to produce young at a pregnancy immediately following a progesterone-treated pregnancy was not impaired.

PIP: Embryo mortality and altered uterine luminal proteins were studied in progesterone-treated rabbits. Progesterone was injected into rabbits on Days -2, -1, and 0 (the day of mating) at doses of .5, 1, and 1 mg, respectively. Normal fertilization rates resulted, however, embryonic death occurred by Day 4. Embryos in progesterone-treated does for up to 3 days survived normally when transferred to normal recipients, whereas Day 4 embryos from treated does exhibited a reduced ability to implant. The uterine fluid (UF) protein pattern was examined on Days 1-7 after mating. Total UF protein levels were significantly greater (p less than .05) in treated animals on Day 4 than in controls. Uteroglobin secretion was significantly advanced (p less than .05) in the treated animals by Day 3. Examination revealed a delay in the time of the arrival of embryos into the uterus in progesterone-treated rabbits. This delay, coupled with the earlier secretion of uteroglobin in the treated- rabbits, suggested a possible asynchrony of approximately 1 day between embryo arrival in the uterus and certain uterine proteins. Embryonic development in vitro and in vivo was examined after exposure to UF collected at various gestational stages. More normal Day 3 morulae placed in UF from Day 3 control and Day 2 progesterone-treated rabbits developed than similar morulae placed in UF from Day 2 controls and Day 3 progesterone-treaded does. Therefore, partial physiologic synchrony was achieved, suggesting that ''asynchronous'' UF can be embryotoxic. It is concluded that the ability of does to produce young at a pregnancy immediately following a progesterone-treated pregnancy was not impaired.

Spermagglutinin titers and their relationship to fertility in isoimmunized female rabbits.

The sera from 48 female rabbits immunized by a series of multiple intradermal injections of washed epididymal, washed ejaculated, and beta-amylase-treated rabbit spermatozoa in complete adjuvant were examined for spermagglutinins by the Kibrick gel agglutination test and a slight modification of the Shulman capillary agglutination test. Control animals receiving the adjuvant or saline usually had no positive titers. All three antigenic preparations produced similar titers, positive at dilutions as high as 8192-fold with Kibrick test and 256-fold with the Shulman test. Maximal titer development was reached 4 to 6 weeks after starting the immunization, and positive sera were obtained from some does for 25 weeks. The correlation coefficients between positive titers obtained by the two tests were r = 0.91 during the first 10 weeks and 0.41 at 15 to 25 weeks after immunization.

Polyacrylamide as a substitute for cervical mucus in sperm migration tests.

A synthetic migration medium for capillary sperm migration studies was developed. Parallel sperm migration in 1.8% polyacrylamide cross-linked with 0.042% N,N'-methylene bis acrylamide was similar to sperm migration in bovine cervical mucus. Bull spermatozoa varying widely in migration distances in bovine cervical mucus maintained similar relative migration distances in this synthetic medium. The advantages of the synthetic medium are its availability in large quantities, its uniformity, and its stability. There was no change in parallel sperm migration distances in the synthetic medium stored at 4 degrees C for up to 4 months. Use of this synthetic medium for human or bovine sperm migration studies would appear to overcome problems associated with the variability of cervical mucus.

Influence of bovine cervical mucus samples and storage conditions on sperm migration in vitro.

In vitro sperm migration assays were performed using bovine spermatozoa and cervical mucus. Experiments were designed to test the effects of storage temperature, method of storage, duration of storage, and source of cervical mucus. Significant variation in migration of spermatozoa was due both to differences in mucus samples and to short-term mucus storage at temperatures ranging from ambient to -196 degrees C. The parallel-orienting effect of cervical mucus on migrating sperm was shown to be a major factor in quantitative assays based upon migration distance. Thus, comparisons of migration among different specimens of semen likely will be biased unless the tests are run simultaneously. Implications of these results are discussed relative to the performance of quantitative sperm migration assays in the clinical or research laboratory.

Migration of fresh and cryopreserved human spermatozoa in polyacrylamide gel.

The ability of freshly collected and frozen human spermatozoa to migrate in round capillary tubes containing specially formulated polyacrylamide gel was investigated, using 33 ejaculates from 27 donors. Each semen sample was divided; one portion was left undiluted, and the other portion was diluted to 50 x 10(6) sperm/ml. Glycerol was used as the cryoprotectant. The percentage of motile sperm cells was determined before and after freezing. Fresh semen contained a higher percentage of motile cells, which migrated farther than those of cryopreserved-thawed semen. Various correlations between the percentage of motile sperm and migration distance ranged from 0.57 to 0.62. There was a low positive correlation of migration distance with sperm cell concentration per milliliter, r = 0.25 to 0.34; and thus adjusting semen samples to a standard sperm concentration improved the accuracy of the test only slightly. The regression coefficient of migration distance on the percentage of motile sperm in fresh semen was 0.65, indicating that for each 10% increase in sperm motility, migration distance is predicted to increase 6.5 mm. Five batches of polyacrylamide gel gave uniform results, and the application of this stable gel to fertility investigations is discussed.

Quantification of bovine sperm separation by a swim-up method. Relationship to sperm motility, integrity of acrosomes, sperm migration in polyacrylamide gel and fertility.

The number of bovine spermatozoa separated in a swim-up procedure was quantified using an electronic cell counter. In an initial test of the swim-up procedure, non-frozen sperm samples with different ratios of live to dead cells were prepared and tested for the number of spermatozoa counted by the swim-up procedure. In ejaculates from six bulls, the number of spermatozoa swimming up was related to the number of live cells present (R2 = 0.97). Next, sperm quality of frozen-thawed semen immediately after thawing was measured at 37 C by swim-up sperm count, sperm motility, spermatozoa with an intact acrosome and migration in polyacrylamide gel and then compared with the fertility of the semen used for artificial insemination. Twenty-nine ejaculates of frozen-thawed semen from 11 bulls were evaluated. Correlations with fertility were highest on an ejaculate basis for motility (r = 0.41, P = 0.05) and for swim-up sperm count (r = 0.35, P = 0.06). On a bull basis, swim-up sperm count had the highest correlation with fertility (r = 0.59, P = 0.06). In a multiple regression model to predict male fertility that included all described measures of semen quality, a R2 value of 0.69 was obtained. This is the first report showing that the ability of spermatozoa to swim out of a more dense medium (whole milk-glycerol extender) into culture media is quantitatively related to in vivo fertility.

High catalase content of rabbit semen appears to be inherited.

Reactive oxygen species, as hydrogen peroxide, can be damaging to sperm. Most species have little protective catalase in their semen, but rabbit semen contains substantial amounts of catalase. The objective of the present study was to characterize a substantial number of Dutch rabbits for seminal content of catalase and determine whether differences were inherited. Usually, 4 or more semen samples were analyzed per male. Catalase was measured by a gasometric procedure. In Experiment 1, the correlation between duplicate determinations was r = .99, and between 2 sets of semen samples from 55 males it was r = .95. There was a significant difference (P < .05) among pairs of males from 6 litters. In Experiment 2, semen from each of 11 males collected at an interval of 1 year contained an average of 13 and 12 units of catalase per mL of semen in consecutive years. The correlation between pairs of samples was r = .85. This indicated that the condition was permanent, and possibly genetically controlled. Experiment 3 analyzed the catalase content of semen from 7 sires and 32 sons. The heritability of seminal catalase concentration was 0.48. These studies indicate that rabbit seminal plasma is high in catalase, and that a substantial portion of the differences among males are under genetic control.

Accessory sperm as an indication of fertilizing ability of rabbit spermatozoa frozen in egg yolk-acetamide with detergent.

Many factors besides initial semen quality affect fertilization rates as sperm interact with the environment of the female reproductive tract. One of these factors is sperm transport, which can be evaluated by accessory sperm counts. Dutch rabbits were used to test the effects on sperm transport, fertilization, and production of young when sodium and triethanolamine lauryl sulfate (STLS) detergent was added to a medium for sperm cryopreservation. When STLS was added in 10 concentrations ranging from 0% to 2.0% (vol/vol) to an egg yolk-acetamide semen extender, optimal post-thaw motility of rabbit sperm occurred when 0.2% to 0.7% STLS was included. However, when 0%, 0.2%, and 0.7% STLS was included to cryopreserve sperm used for insemination, the fertilization rates were 95%, 68%, and 75%, and the corresponding mean numbers of accessory sperm per embryo were 13.1, 1.7, and 0.4 (P < .05). In another experiment, increasing the acetamide concentration from 0.75 M to 1.25 M decreased fertilization rates from 66% to 35%, and was associated with 4.5 and 0.6 accessory sperm per embryo (P < .05). In the final experiment, 48 does inseminated with sperm cryopreserved with 0%, 0.35%, and 0.70% STLS were allowed to produce young. Corresponding pregnancy rates were 56%, 56%, and 31% (P < .05), and litter sizes were 5.6, 4.1, and 4.2 (P > .05). In these studies, low concentrations of STLS improved motility of frozen-thawed sperm, but fertilization and pregnancy rates were reduced. Sperm transport was correspondingly reduced, and the accessory sperm count provided a reliable measure of the effect of STLS on fertility in contrast to the assessment of the percentage of motile sperm.

Effect of repeated testicular biopsy on testis function and semen quality in dogs.

The study was designed to quantitatively evaluate the effect of repeated testicular biopsy of avascular areas by the open method on spermatogenesis, semen quality, and sperm output in dogs. After a 5-week standardization period of semen collections 3x per week, 10 sexually mature Beagle dogs were divided into two groups, approximately equalized based on previous sperm output. Semen collections were continued for 20 weeks. One group was controls without treatment until unilaterally orchidectomized after weeks 16 and 20; a second group had one testis biopsied on weeks 1, 5, and 9 and removed after week 16, with the second testis removed after week 20. Sperm output per week during the summer declined in the control group from 851 x 10(6) to 725 x 10(6) (15%). The decline in the biopsied control group collected simultaneously was 22%, presumably reflecting a seasonal effect plus a possible small treatment effect. At 16 weeks, the testes removed from the controls averaged 7.1 g and the testes biopsied three times in the second group averaged 6.4 g (P > 0.05). Scrotal-testicular measurements taken in live animals were correlated with testicular weights at orchidectomy (r = 0.81). An average of 36.7 mg of tissue was removed at each biopsy, sufficient to evaluate 100 to >200 seminiferous tubules in cross-section. Sperm motility and frequency of all stages of the cycle of the seminiferous epithelium were similar between control and biopsied dogs. It is concluded that repeated testicular biopsy of avascular areas can provide sufficient tissue for histopathologic evaluation without significant interference with spermatogenesis.

Computerized imaging and scanning electron microscope (SEM) analysis of co-cultured fresh and frozen bovine sperm.

Fresh and frozen-thawed bull sperm were incubated with bovine oviductal epithelial cells and segments from the oviducts to examine the usefulness of these culture systems to model sperm changes in vivo. Changes in sperm motion characteristics [computer-assisted sperm analysis (CASA)] and surface morphology [scanning electron microscopy (SEM)] were evaluated. In Experiment 1, fresh and frozen sperm were suspended in the Brackett and Oliphant medium or modified Tyrodes medium (mTALP) and incubated for 0, 3, 6, and 9 hours in direct contact with bovine oviductal-epithelial cell (BOEC) monolayers prepared from oviducts of cows in the periovulatory phase of estrus. The percentage of motile sperm decreased gradually in mTALP, but decreased rapidly in Brackett's defined medium after 3 hours of incubation, with overall averages of 55 and 32%, respectively. The percentage of motile fresh sperm exceeded frozen-thawed sperm under all conditions. In Experiment 2, sperm suspended with mTALP were incubated in dishes without monolayers (control), with monolayers, and within the segments of the oviduct for 0, 3, and 6 hours. In the epithelial cell monolayers, the percentage of motile sperm was similar to the controls throughout incubation, but after 3 hours in the oviductal segments, a decrease, partly associated with more rapid rupture of acrosomal membranes occurred. Sperm velocity was higher (100 microns/second) in fresh sperm than in frozen sperm (85 microns/second). Acrosomal changes, discernible with SEM after 3 hours of incubation, increased with time and were always found more often in frozen than in fresh sperm. The BOEC-monolayer system provided a useful in vitro model to study pre-fertilization changes in sperm.

A comparison of two computer-automated semen analysis instruments for the evaluation of sperm motion characteristics in the stallion.

Two commercially available computer-automate semen analysis instruments (CellSoft Automated Semen Analyzer and HTM-2000 Motion Analyzer) were compared for their ability to report similar results based on the analysis of pre-recorded video tapes of extended, motile stallion semen. The determinations of the percentage of motile cells by these instruments were more similar than the comparisons between subjective estimates and either instrument. However, mean values obtained from the same sample may still differ by as much as 30 percentage units between instruments. Instruments varied with regard to the determinations of mean sperm curvilinear velocity and sperm concentration, but mean sperm linearity determinations were similar between the instruments. We concluded that the determinations of sperm motion characteristics by subjective estimation, CellSoft Automated Semen Analyzer, and HTM-2000 Motility Analyzer are often dissimilar, making direct comparisons of results difficult.