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D E Boatman

Publications and source records attributed to D E Boatman.

14 recordsLinked to original sources
PubMed1995

Identification of a sperm penetration factor in the oviduct of the golden hamster.

Previously, we found oviductal eggs to be significantly more penetrable and fertilizable in vitro than ovulated eggs collected from the ovarian bursa, while bursal eggs were comparable to mature (unovulated) follicular eggs. Incubation of follicular eggs with a soluble eluate of oviductal egg cumulus complexes (COF) increased sperm penetration: the activity was macromolecular, was destroyed at 56 degrees C, and was produced in the oviduct. We now report purification of this oviductal factor that enhances penetration of follicular eggs and have identified it as oviductin (OVN). Oviducts, 1-1.5 h post-LH from eCG-primed females, were homogenized and the cytosolic fraction was chromatographed on a Helix pomatia lectin affinity column; specific proteins were eluted with 0.2 M N-acetyl-D-galactosamine. Fractions were monitored by dot-blot assay using as the primary antibody monoclonal antibody (mAb) 1C4 against OVN. Proteins were resolved by one-dimensional SDS-gel electrophoresis, followed by electrotransfer and immunostaining of Western blots. OVN fractions were indexed to COF by quantitative dot-blot assay, and activity was bioassayed by penetration of follicular eggs within 1 h of coincubation with precapacitated sperm +/- factors: COF and BSA (high and low controls, respectively) and fractions from the lectin-isolated peak. The mean penetration rates for three isolations were 17 +/- 4.0a, 51.7 +/- 5.0b, and 49 +/- 2.7b% for BSA, COF, and column fractions, respectively (p < or = 0.05). Purified OVN bound to follicular zonae during culture. Acrosome-intact sperm heads bound OVN during 30 min of incubation both before (t = 0 h) and after capacitation (t = 5.5 h) (visualized by indirect immunofluorescence).(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗
PubMed1995

Progesterone concentrations in serum, follicular fluid, and oviductal fluid of the golden hamster during the periovulatory period.

In vitro, progesterone induces capacitation, hyperactivated motility, and acrosome reactions in sperm, possibly acting through a non-genomic plasma membrane receptor, but neither progesterone functions nor concentration within the mammalian oviduct during the periovulatory period is well characterized. The objectives of this study were to determine the physiological concentrations of progesterone in serum, follicular fluid, and oviductal fluid of golden hamsters during the period of the reproductive cycle in which capacitation and fertilization occur. Fluids were collected from different groups of animals for each of four time points: the normal time for mating (5-6 h post-LH surge [post-LH]), early in capacitation (8-9 h post-LH), immediately preovulation (11-12 h post-LH), and after ovulation near the beginning of fertilization (14-15 h post-LH). Oviductal fluid was collected by 1-h cannulation followed by cardiac puncture for serum collection and follicle aspiration for follicular fluid. Within each time period, the three types of fluid differed significantly in progesterone concentration. Over time, concentrations of progesterone did not change in either serum (range: 5.64-12.85 ng/ml) or follicular fluid (range: 4.2-7.4 micrograms/ml), but the concentration of progesterone in oviductal fluid decreased from 175.06 ng/ml at the first period to 44.01 ng/ml at the fourth (p < 0.05), while the volume of oviductal secretions collected by the same sampling procedure increased from 1.6 to 2.9 microliters (p < 0.05). With this information concerning in vivo concentrations of progesterone during capacitation and fertilization, the physiological role of progesterone in sperm-egg interactions can be addressed.

Animals↗
PubMed1995

Effects of progesterone on in vitro sperm capacitation and egg penetration in the golden hamster.

Progesterone (P) is postulated to have a physiological role in vivo during sperm capacitation and/or during sperm-egg interaction as a cofactor for induction of the acrosome reaction. Effects on in vitro fertilization were tested by adding P (0.01-2 micrograms/ml) to sperm during capacitation (4-6 h) or after capacitation during sperm-egg interaction for 1 h. Additionally, to test for acceleration of the onset of capacitation by P, eggs were inseminated with sperm that were incubated with P for 3.5 and 5 h. Neither the solvent dimethyl sulfoxide nor P affected the motility or vigor of treated sperm. The effects of P on egg penetration were dependent on the dose, the temporal sequence of its addition to sperm (i.e., before or after capacitation), and the duration of sperm exposure. Capacitation of sperm with 20 ng/ml P for 5 h, but not 3.5 h, increased the percentage of eggs penetrated by sperm over controls (52.65 +/- 4.01% vs. 39.30 +/- 5.18%, p < 0.05). But P (0.01-2 micrograms/ml), either added after capacitation (4.5 h) during sperm-egg coincubation (1 h) only, or present (1 micrograms/ml) throughout capacitation (4-6 h) and sperm-egg coincubation (1 h), did not increase egg penetration over control levels. In conclusion, the best results occurred with a protocol that mimicked in vivo conditions of capacitation time (5 h) and preovulatory oviductal levels of P (20 ng/ml).

Animals↗
PubMed1991

Detection of a soluble acrosome reaction-inducing factor, different from serum albumin, associated with the ovulated egg-cumulus complex.

Soluble extracts of the ovulated hamster egg-cumulus complex (ECC) were tested on capacitated sperm for activity in inducing the physiological acrosome reaction (AR). Evidence for occurrence of the physiological AR included enhanced sperm penetration of intact homologous zonae pellucidae as well as induction of AR in nonattached and in zona-bound sperm following a brief coincubation with test compound. Since hamster serum albumin, a major protein of hamster body fluids, also induces spontaneous ARs under certain conditions, it was used as one of the comparators for the acrosome reaction inducing factor (ARIF; Westrick et al., Biol Reprod 32 [Suppl 1]. 213, 1985) activity in the ECC. Sperm exposure to concentrations of the soluble ECC extract ranging from 0.04 to 0.2 mg protein/ml significantly increased penetration of salt-stored zonae by 36%, mean numbers of penetrating sperm by 90%, ARs in nonattached sperm by 65%, and ARs in zona-bound sperm by 102%. Hamster serum albumin added after completion of capacitation had no significant effect on these parameters. We conclude that 1) the ovulated ECC contains a soluble ARIF that augments zona-induced ARs and sperm penetration and 2) the ARIF is not serum albumin.

Acrosome↗
PubMed1991

Bicarbonate: carbon-dioxide regulation of sperm capacitation, hyperactivated motility, and acrosome reactions.

The bicarbonate: CO2 (HCO3-:CO2) concentration dependencies of hamster sperm motility, spontaneous acrosome reactions, and zona penetration (used to assay the zona-induced acrosome reaction) were examined. A cross-over experimental design was used to segregate effects on early stages of capacitation, spanning the first 5 h of incubation, from those on acrosome reactions and zona penetration during the last 1 h. After 5 h, HCO3-:CO2 concentrations were increased, decreased, or kept the same for 1 h. Compared to no HCO3-:CO2, as little as 2.9 mM: 0.6% HCO3-:CO2 increased the sperm motility index (MI) by 2.7-3.6 times. When HCO3-:CO2 was continuously present, both progressive and hyperactivated motility were stimulated by HCO3-:CO2 in a dose-dependent manner by 3-4 h, well before completion of capacitation. Stimulation of acrosome reactions or zona penetration, by addition of HCO3-:CO2 to sperm for 1 h late in capacitation, depended mainly on levels of HCO3-:CO2 present earlier in capacitation. When 25 mM: 5% HCO3-:CO2 was added only at 5 h, responses were significantly lower than with sperm treated continuously with the same concentration of HCO3-:CO2, being 2.5 times lower for MI, 2 times lower for acrosome reactions, and 6.3 times lower for zona penetration. In contrast, decreasing HCO3-:CO2 to suboptimal levels after 5 h did not decrease any 6-h sperm responses significantly. The average maximal and one-half maximal preincubation HCO3- concentrations for all responses were 34.2 +/- 1.0 and 9.2 +/- 0.3 mM, respectively. Zona penetration and hyperactivation were highly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗
PubMed1990

Relationships between follicular fluid steroid hormone concentrations, oocyte maturity, in vitro fertilization and embryonic development in the rhesus monkey.

Oocytes and matched samples of follicular fluid (FF) were obtained from 70 follicles of five rhesus monkeys stimulated with either pregnant mare serum gonadotropin or human menopausal gonadotropin. Follicular aspiration was performed 30-32 h after human chorionic gonadotropin administration. The concentrations of estradiol (E2), progesterone (P), testosterone (T), and dihydrotestosterone (DHT) in FF were measured. Twenty-six percent of oocytes were classified as mature (M), 41% matured in vitro (Miv), 13% were dysmature, and 20% atretic. M oocytes were associated with significantly higher levels of P and a higher P:E2 ratio. There were no differences in hormone levels associated with fertilized and nonfertilized oocytes. Thirty-five embryos developed to the six- to eight-cell stage in vitro, of which 13 exhibited optimal cleavage rates. Significantly lower levels of E2 and higher P:E2 ratios were associated with the more rapidly cleaving embryos. Proportionally more embryos showing optimal cleavage rates developed from M compared to Miv oocytes, and only embryos derived from M oocytes developed to blastocysts in culture. Optimal cleavage rates to the six- to eight-cell stage in vitro, rather than fertilization rates, are a better indicator of (subsequent) developmental capacity, and, in this study, embryonic development was closely associated with the maturity of the oocyte at recovery.

Animals↗
PubMed1988

A quantitative assay for capacitation: evaluation of multiple sperm penetration through the zona pellucida of salt-stored hamster eggs.

The endpoint for sperm capacitation occurs when spermatozoa become able to penetrate intact zonae pellucidae of unfertilized homologous eggs. Activation of eggs stimulated by sperm fusion or by gamete aging initiates changes in zonae pellucidae that bar further sperm entry. This zona block mechanism reduces the usefulness of eggs as indicators for sperm capacitation. Egg storage in concentrated salt solutions destroys the zona block mechanism while retaining the biological sperm receptor/activator functions of the zona pellucida [Yanagimachi et al., Fertil Steril 31:562-574, 1979]. We have developed techniques for the quantitative assay of capacitation using multiple sperm penetration into zonae and have evaluated the sperm-response characteristics of hamster eggs stored in a modified salt solution (STOR). Sperm, preincubated in capacitating or noncapacitating treatments (CAP or NOCAP, respectively), were coincubated with unfertilized and fertilized STOR-treated zonae for 4 hr. Then zonae were stripped of externally bound sperm and the sperm heads in the perivitelline space (PVS) were fluorescently labeled with Hoechst dye 33342. Entry of CAP sperm into the PVS of STOR-treated unfertilized eggs was highly correlated with sperm concentration (Rw = .859) and was log linear from 1 X 10(3)-2 X 10(5) CAP sperm/ml. At 2 X 10(5) CAP sperm/ml, the mean number of PVS sperm was 63.5 and the maximum observed was 158. NOCAP sperm very rarely penetrated unfertilized zonae (2 sperm into 75 eggs). Few CAP sperm entered the PVS of STOR-treated fertilized eggs (two sperm into 54 eggs at 2 X 10(5) sperm/ml) unless the sperm concentration was raised to high levels. Differences between replicates were due to male but not female (ie, egg) differences. We conclude that 1) STOR-treated unfertilized zonae can be used to accurately quantitate differences between sperm capacitation and/or fertility states and 2) the availability of large numbers of homogeneous "shelf-stable" zonae will make it feasible to perform hamster sperm capacitation bioassays on a large scale.

Animals↗
PubMed1984

Birth of rhesus monkey infant after in vitro fertilization and nonsurgical embryo transfer.

The birth of a rhesus monkey resulting from in vitro fertilization is reported. Oocytes recovered at laparoscopy from five gonadotropin-stimulated donors were inseminated in vitro with sperm preincubated with caffeine and dibutyryl cyclic AMP. After insemination, oocytes were cultured for 33-46 hr. Twenty-two embryos were transferred nonsurgically into 11 recipient females. One recipient showed signs of implantation but did not carry to term. A second female became pregnant after receiving one 4-cell and one 6-cell embryo fertilized in vitro. The subsequent course of early pregnancy and embryonic and fetal development were characteristic of normal singleton pregnancies. A healthy term male infant was delivered by Cesarean section 176 days after fertilization. This birth has validated our procedures for in vitro fertilization of rhesus monkey gametes and provides an experimental model for studies of early embryonic development in primates.

Animals↗
PubMed1984

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators.

Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.

Animals↗
PubMed1980

An enzymatically driven membrane reconstitution from solubilized components.

Acyltransferase activity is present in a variety of membranes species, including liver microsomes. The substrates of this enzyme are lysophosphatides and acyl CoA derivatives. We have found that the detergent effect of these substrates can be used to solubilize rat liver microsomes. If the solubilized fraction in incubated, the acyltransferase acylates the lysophosphatide and thereby degrades the detergent effect so that vesicular membranes re-form. Gel electrophoresis patterns show that the reconstituted membranes contain all of the major protein components of the original microsomes. A marker enzyme for liver microsomes, NADPH-cytochrome c reductase, was present in the reconstituted membranes at 70% of the specific activity in the original microsomes, and freeze-fracture electron microscopy showed intramembrane particles on all fracture faces. The system may provide a useful model for studies particles on all fracture faces. This system may provide a useful model for studies of certain membrane biogenesis reactions that utilize acyltransferase in vivo.

Acyl Coenzyme A↗
PubMedDate not supplied

Addition of hypotaurine can reactivate immotile golden hamster spermatozoa.

Hamster epididymal spermatozoa became virtually immotile following washing and dilution in chemically defined medium (TLP-PVA). The sperm motility factors (penicillamine, hypotaurine, and epinephrine: PHE) were examined for their ability to reactivate immotile sperm. Sperm could be reactivated by addition of PHE at 1 h of incubation. Hypotaurine alone was capable of reactivating sperm motility, but epinephrine and penicillamine together were not. However, overall sperm motility and percentage of motile sperm during incubation were higher when PHE components were used in combination than when hypotaurine was used alone. Addition of hypotaurine to immotile sperm suspensions could be postponed for up to 6 h with subsequent recovery of sperm motility, although the degree of recovery of motility declined progressively with each hour that addition of hypotaurine was delayed. The rescuing effect of hypotaurine was due to an increase both in the percentage of motile sperm and in the quality (grade) of sperm motility. The data show that hypotaurine is required for expression of sperm motility in the hamster, and support the concept that the loss of hypotaurine from sperm following washing and dilution is responsible for the sperm-immobilizing effect of these procedures. Additionally, the data demonstrate that hamster sperm can remain viable for several hours after becoming immotile, and that many of the immotile sperm are capable of being reactivated.

Animals↗