Four orchidectomized rhesus monkeys (3-3.5 yr of age) were treated for 62 days with daily i.m. injections of hydrocortisone acetate (HCA) at a dose of 10-20 mg/(kg BW X day), and blood samples were obtained daily or every other day before, during, and after treatment. Hydrocortisone acetate injections resulted in a progressive rise in mean plasma cortisol from basal concentrations of 17-35 micrograms/100 ml prior to initiation of steroid treatment to approximately 150 micrograms/100 ml 5 wk later. When serum cortisol concentrations reached 100 micrograms/100 ml, 3-4 wk after the initiation of HCA treatment, circulating luteinizing hormone (LH) and follicle-stimulating hormone (FSH) began to decline, reaching nondetectable concentrations 35 days later. Withdrawal of HCA resulted in a return in plasma cortisol concentrations to pretreatment control levels, which was associated with a complete restoration of gonadotropin secretion. In 2 animals, administration of an intermittent i.v. infusion of gonadotropin-releasing hormone (GnRH) (0.1 micrograms/min for 3 min once every hour), which appears to stimulate the gonadotropes in a physiologic manner, reversed the cortisol-induced inhibition of gonadotropin secretion, restoring circulating LH and FSH concentrations to within 80-100% of control. These results suggest that, in the rhesus monkey, the major site of the inhibitory action of cortisol on gonadotropin release resides at a suprapituitary level and is mediated by interruption of hypothalamic GnRH release.
The concentrations of hypoxanthine and adenosine in ovarian follicular fluid were estimated, using high-performance liquid chromatography, for three groups of mice: 1) pregnant mare's serum gonadotropin (PMSG)-primed mice; 2) PMSG-primed mice 2 h after injection with human chorionic gonadotropin (hCG); and 3) PMSG-primed mice 5 h after injection with hCG. The concentration of hypoxanthine in follicular fluid of Group 1 mice was 2-4 mM and of adenosine was 0.35-0.70 mM. There was no difference in the concentrations of these purines in the follicular fluid of Group 2 mice, in which maturation had been induced with hCG but the samples were taken just before germinal vesicle breakdown (GVBD). Therefore, a decrease in the concentrations of these purines does not appear to induce GVBD. A significant decrease in the concentrations of hypoxanthine and adenosine was observed in the follicular fluid of Group 3 mice in which GVBD had already occurred. This decrease was probably a result of an increase in follicular fluid volume. Adenosine had a significant, but transient, effect in maintaining both cumulus cell-enclosed and denuded oocytes in meiotic arrest; all oocytes had undergone GVBD by 100 min incubation in 1 mM adenosine. When GVBD was assessed after 3 h culture, concentrations up to 5 mM adenosine failed to maintain meiotic arrest. In contrast, hypoxanthine (2-5 mM) had a dose-dependent effect in maintaining both cumulus cell-enclosed and denuded oocytes in meiotic arrest that was sustained up to 24 h. Cumulus cell-enclosed oocytes were always more sensitive to hypoxanthine than were denuded oocytes. There was a strong synergistic effect of adenosine and hypoxanthine in maintaining meiotic arrest; 4 mM hypoxanthine and 0.75 mM adenosine maintained more than 95% of the oocytes in meiotic arrest for culture periods up to 24 h. This action was completely reversible by withdrawal of the purines. It is hypothesized that the synergistic effect of these purines may result both by promoting cyclic adenosine monophosphate synthesis (adenosine), and by preventing its hydrolysis (hypoxanthine).
The role of albumin in mouse sperm capacitation was studied in relation to its activities as a lipid-solubilizing protein and a sterol acceptor. Two bovine serum albumins (BSA) which supported capacitation, Fraction V and fatty acid-free, both contained cholesterol and phospholipid but were without detectable levels of serum high-density lipoprotein (HDL). The lipid content of BSA could be reduced by trichloroacetic acid (TCA) precipitation; however, removal of all detectable lipids required precipitation with ethanolic acetone and diethyl ether extraction. In medium supplemented with Fraction V, fatty acid-free, or TCA-precipitated BSA, mouse sperm were capacitated as evidenced by their ability to fertilize eggs, concomitant with decreases in total cellular sterol and increases in phospholipid content. Delipidated BSA, fractionated on Sephadex G-100 in guanidine HCl also supported capacitation and mediated a 20% decrease in sperm sterol content, while cellular phospholipid levels remained unchanged. When BSA was modified by cholesterol augmentation, fertilization was inhibited in a cholesterol dose-dependent manner. These findings suggest that modulation of sperm lipid levels comprises an event of capacitation and that albumin mediates this process through its activity as a sterol acceptor.
Ram spermatozoa were obtained from different regions (caput, corpus, and cauda) of the epididymis and their plasma membrane was removed using a nitrogen cavitation treatment (750 psi, 10 min equilibration at 4 degrees C). Membrane was recovered after sucrose gradient centrifugation and identified using 125I-succinylated concanavalin A (125I-succConA) as a surface marker. Based on fluorescein isothiocyanate-succConA (FITC-succConA) labeling and electron microscopy, cavitation removed plasma membrane from the anterior sperm head in the area overlying the acrosome. Cholesterol was the major sterol in plasma membrane, with desmosterol present in sperm entering the epididymis (caput sperm) but negligible in sperm after epididymal transit (cauda sperm). Ethanolamine and choline phosphoglycerides represented 70-80% of membrane phospholipids, with the ethanolamine fraction decreasing relative to choline phosphoglycerides during epididymal transit. The molar ratio of cholesterol to phospholipid increased in the plasma membrane during maturation. The bulk phospholipid-bound fatty acids consisted primarily of palmitoyl acyl groups (16:0) in caput sperm and docosahexaenoyl acyl groups (22:6) in cauda sperm. The choline phosphoglyceride fraction was purified and analyzed. It consisted of a mixture of ether acyl glycero-3-phosphocholine and diacyl phosphoglyceride, with the dominant acyl residue, at all stages of epididymal maturation, being 22:6 throughout epididymal transit. The significance of these findings relative to acquisition of fertilization capacity by sperm during epididymal maturation is discussed.
Bovine conceptuses from Days 16 (n = 4), 19 (n = 6), 22 (n = 3), and 24 (n = 4), and chorion from Day 69 (estrus/mating = Day 0) were cultured for 24 h in modified minimum essential medium (MEM) in the presence of radioactive L-leucine [( 3H] leucine) to characterize de novo synthesis and release of proteins. Proteins released into MEM were identified by two-dimensional polyacrylamide gel electrophoresis, fluorography, and gel and ion exchange chromatography. Major polypeptides identified in MEM were different from those identified in conceptus and chorionic tissues. Both uptake of [3H] leucine and quality of polypeptides produced de novo and released into MEM were related to stage of conceptus development. Percent retention of [3H] leucine in MEM was lowest (P less than 0.01) in Day 16 cultures (1.2 +/- 4.1%), increased in Days 19 (16.8 +/- 3.7%) and 22 cultures (20.9 +/- 5.8%), and decreased (P less than 0.07) in Day 24 cultures (6.9 +/- 4.1%). Complexity of polypeptides increased after Day 16. Days 16, 19, 22 and 24 conceptus culture MEM was enriched in low-Mr, acidic polypeptides (Mr/isoelectric point ranges: 22K-26K/6.5-5.6, 20K-26K/5.5-5.4, and 16K-20K/5.0-4.5), which were not prominent products of Day 29 and 69 tissues. A high-Mr (Mr +/- SEM; 735K +/- 22K) glycoprotein was produced by all conceptus and chorionic tissues. The transient nature of production of low-Mr polypeptides suggests that they may be required during the periattachment period.
Previous studies in which prostaglandin (PG) production was inhibited for a limited time by the s.c. administration of indomethacin have suggested that PGs are involved in the initiation of decidualization as well as the growth and differentiation of decidual cells. To reduce PG production during decidualization, in the present study indomethacin was infused from Alzet osmotic minipumps into the uterine lumen of ovariectomized rats with uteri sensitized for decidualization. To determine the effect of route of indomethacin administration on decidualization, rats received a single s.c. injection of indomethacin or its vehicle, and unilateral intrauterine infusion of indomethacin or its vehicle, in a factorial experiment. The inhibitory effects on decidualization, as assessed 5 days later by uterine weights, were greatest when both treatments were combined. Prostaglandins E and F concentrations 24 and 48 h after the insertion of the pumps were lower in the indomethacin-infused horns, suggesting that the indomethacin reduced uterine PG production. By contrast, subcutaneously administered indomethacin reduced uterine PG concentrations at 24 h but not at 48 h. Prostaglandin E2 and PGF2 alpha alone or combined, infused with indomethacin into the uterine lumen of rats treated subcutaneously with indomethacin, overrode the inhibitory effects of indomethacin. The dose-response relationships between these PGs and decidualization did not differ. These data suggest that PGs are required during the growth and differentiation of decidual cells from endometrial stromal cells.
The concentrations of six steroids and of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in follicular fluid from preovulatory and large atretic follicles of normal Holstein heifers and from preovulatory follicles of heifers treated with a hormonal regimen that induces superovulation. Follicular fluid from preovulatory follicles of normal animals obtained prior to the LH surge contained extremely high concentrations of estradiol (1.1 +/- 0.06 micrograms/ml), with estrone concentrations about 20-fold less. Androstenedione was the predominant aromatizable androgen (278 +/- 44 ng/ml; testosterone = 150 +/- 39 ng/ml). Pregnenolone (40 +/- 3 ng/ml) was consistently higher than progesterone (25 +/- 3 ng/ml). In fluid obtained at 15 and 24 h after the onset of estrus, estradiol concentrations had declined 6- and 12-fold, respectively; androgen concentrations had decreased 10- to 20-fold; and progesterone concentrations were increased, whereas pregnenolone concentrations had declined. Concentrations of LH and FSH in these follicles were similar to plasma levels of these hormones before and after the gonadotropin surges. The most striking difference between mean steroid levels in large atretic follicles (greater than 1 cm in diameter) and preovulatory follicles obtained before the LH surge was that estradiol concentrations were about 150 times lower in atretic follicles. Atretic follicles also had much lower concentrations of LH and slightly lower concentrations of FSH than preovulatory follicles. Hormone concentrations in follicles obtained at 12 h after the onset of estrus from heifers primed for superovulation were similar to those observed in normal preovulatory follicles at estrus + 15 h, except that estrogen concentrations were about 6-40 times lower and there was more variability among animals for both steroid and gonadotropin concentrations. Variability in the concentrations of reproductive hormones in fluid from heifers primed for superovulation suggests that the variations in numbers of normal embryos obtained with this treatment may be due, at least in part, to abnormal follicular steroidogenesis.
Environmental exposure to toxic levels of lead occurs in a number of industries with potential adverse effects on the reproductive capacity of exposed men. Clinical and animal studies indicate that abnormalities of spermatogenesis result from toxic lead exposure, but the pathogenetic mechanisms involved have not been identified. In order to ascertain what reproductive abnormalities occur in experimental animals when exposed to low levels of lead, 52-day-old animals were treated with water containing 0.0% (control), 0.1%, or 0.3% lead acetate for 30 days prior to killing. Whole blood serum lead levels were below detection (less than 7 micrograms/dl) in the control animals, 34 +/- 3 micrograms/dl in the 0.1% group, and 60 +/- 4 micrograms/dl in the 0.3% group (P less than 0.001). Significant negative correlations between whole blood lead levels and serum and intratesticular testosterone values were found (r = 0.64, P less than 0.001 and r = 0.6, P less than 0.001, respectively). As the level of lead exposure increased, intratesticular sperm counts significantly decreased (r = 0.81, P less than 0.001). No significant changes in serum luteinizing hormone (LH) values were found, but sperm follicle-stimulating hormone (FSH) values were significantly suppressed (P less than 0.05) after lead treatment. There was a significant decrease in ventral prostate weight (P less than 0.05), but no differences in testicular or seminal vesicle weights. Our data indicate that dietary exposure to lead resulting in whole blood serum lead values considered acceptable in the workplace (less than or equal to 40 micrograms/dl) causes inhibition of testicular function.
Restricting the food intake of female mice by alternating days of feeding and fasting delayed the age-related loss of estrous cycling potential and retarded the rate of follicular depletion, as determined after reinstatement of ad libitum (AL) feeding. During the period of food restriction (FR; 3.5-10.5 mo), food intake and body weight were about 80% of AL values. Mice were acyclic and predominantly in a state of diestrus during FR, but after reinstatement of an AL diet at 10.5 mo all FR mice resumed cycling regularly. By contrast, 80% of AL controls had become acyclic by this age, and the cycles of the remaining mice were significantly longer than those of the reinstated FR mice. Follicular reserves of 12.5-mo-old FR mice were twice those of age-matched AL controls. Cycling performance of reinstated FR mice, measured by cycle length and the proportion of mice still cycling, was equivalent to that of AL mice when the latter were 2-5 mo younger. Ovarian age, measured by the size of the follicular reserve, was similarly retarded in FR mice. Based on these data and previous evidence that follicular depletion plays a major role in the cessation of cyclicity in this strain, we hypothesize that the delayed loss of estrous cyclicity in aging FR mice is mediated at least in part by the retarding effect of dietary restriction on the rate of follicular depletion.
We have reexamined the possibility that cumulus cell cAMP can enter the oocyte via the gap junctions connecting the two cell types (Schultz et al., 1983a). Since our recent results indicate that the mouse oocyte possesses a very active cyclic nucleotide phosphodiesterase (PDE) (Bornslaeger et al., 1984), we have altered our experimental protocol to ensure that mouse oocyte PDE activity is inhibited throughout the duration of an experiment. Our results demonstrate the apparent transfer of cAMP from cumulus cells to the oocyte; these results are discussed in terms of current models for regulation of mammalian oocyte maturation.
Monoclonal antibodies (mAbs) have been generated to determine the sperm components responsible for interaction with an egg that results in fertilization. Here, we report upon a group of six different mAbs, all of which localize to a restricted region of the sperm head, the equatorial segment. Several of these mAbs demonstrated cross-reactivity with sperm from the other species tested (human, hamster, rabbit); when cross-reaction occurred, the mAb distribution was restricted to the equatorial segment despite the various configurations that this homologous region assumes in different species. When tested for an effect upon the fertilization process in vitro, ascites fluids containing two of the six mAbs, M29 and M37, displayed significant inhibition. The concentration dependency of this inhibition was observed using purified M29 immunoglobulin M, over a range of 0 to 0.2 mg/ml. The mAb inhibition of fertilization was independent of the presence of either the cellular (the cumulus) or acellular (the zona pellucida) layers surrounding the egg, indicating that the specific locus of inhibition for both of these antisperm mAbs was the egg plasma membrane. Immunologic detection of sperm components separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels followed by transfer to nitrocellulose sheets was used to identify the sperm components recognized by two of the mAbs in this group: M29, which inhibited fertilization, and M2, which did not inhibit fertilization. Using M29 mAb, a single sperm component with an apparent subunit molecular weight of approximately 40,000 was detected, whereas in the nitrocellulose strips incubated with M2 mAb two components displayed reactivity, a very prominent band at approximately 44,000 and a tight cluster of bands at approximately 36,000. Parallel nitrocellulose strips of mouse liver did not display these reactivities, consistent with indirect immunofluorescence data in which only testis and sperm, and not liver, kidney, ovary, and epididymal epithelium, demonstrated positive reactivity. These results indicate that the use of mAbs permits identification of sperm components that participate, putatively, in individual events of the fertilization process. Furthermore, using this strategy, we have identified a specific sperm component that appears to be a candidate for a role in sperm fusion with the egg plasma membrane.
Effects of ethane dimethyl sulfonate (EDS) on Leydig cells have been studied using the following parameters: morphology, histochemistry of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and esterase, quantitative activity of esterase, testosterone concentrations in plasma, and steroid production by isolated interstitial cells in vitro. Degenerating Leydig cells were observed within 16 h after the injection of mature rats with EDS (75 mg/kg body weight). At that time the testosterone concentration in plasma and the specific activity of esterase in testis tissue were decreased to approximately 35% and 60% of the control value, respectively. At 48 h after EDS only a few normal Leydig cells were left and the plasma testosterone concentration was less than 5% of the control value. The specific activity of esterase in total testis tissue was similar to the activity of dissected tubules from untreated rats. At 72 h no Leydig cells could be detected and no 3 beta-HSD and esterase-positive cells were present. At that time macrophages were still present in the interstitium and the appearance of the spermatogenic epithelium was normal, but 1 wk after EDS the elongation of spermatids was disturbed, probably due to a lack of testosterone. In some of the animals the cytotoxic effects of EDS on Leydig cells could be partly inhibited by human chorionic gonadotropin treatment. The basal steroid production by interstitial cells from mature rats 72 h after EDS was not significant and no stimulation by LH was observed, whereas no effect of EDS could be detected on steroid production by interstitial cells isolated from immature rats and mice 72 h after treatment. Other compounds with similar structures, such as butane dimethyl sulfonate (busulfan) and ethane methyl sulfonate (EMS) had no effect on Leydig cells from mature rats. It is concluded that EDS specifically destroys Leydig cells in mature rats.
The obscured pronuclei or nuclei in living one- and two-celled pig ova were revealed after centrifugation for 3 min at 15,000 X g. To determine viability of centrifuged ova, one- and two-celled pig ova were collected from superovulated gilts; half of the ova were centrifuged and all ova were transferred into recipient gilts. Prior to transfer all embryos were stained with tetramethylrhodamine isothiocyanate (TRITC) to distinguish the experimental embryos from the recipients's own ova. Centrifuged ova were transferred into one oviduct of recipient gilts and uncentrifuged ova were deposited into the opposite oviduct. Embryos were recovered 4 days after transfer and were stained with lacmoid or Hoechst 33342 to assess their stage of development. Centrifugation had no detectable influence on survival of the recovered embryos to 4 days. Centrifugation is a simple, reliable method for revealing pronuclei and nuclei of one- and two-celled pig ova and apparently does not alter subsequent preimplantation development.
CF-1 female mice were subjected to 24 or 48 h of food deprivation beginning when they were in estrus or diestrus, or when they were 2 or 12 days pregnant, or on Days 2 or 12 of lactation. Ovulation was delayed by a week or more when 48 h of food deprivation was initiated when the female was in diestrus; lesser delays occurred when food deprivation began in estrus. There was little effect of acute food deprivation on pregnancy. Most females deprived of food beginning on Day 2 of lactation ate their young, but females deprived on Day 12 of lactation rarely did so. These results are discussed in terms of the complexity of interacting factors that determine the degree to which each stage of the female's reproductive cycle is susceptible to disruption by acute food deprivation.
To determine the changes in patterns of 17 beta-estradiol and progesterone levels underlying abnormal cycles in bitches immunized with solubilized crude porcine zonae pellucidae (cPZP), to attempt to circumvent these problems by immunizing with a purified zona fraction (pPZP), and to test the effectiveness of different adjuvants, bitches were immunized with cPZP or pPZP 2-6 times with no adjuvant, Freund's adjuvant, alum adjuvant, or the adjuvant CP-20,961. The bitch immunized without adjuvant had a low titer with a normal cycle and fertility. Immunization with cPZP and adjuvant produced moderate to high titers of antizona antibodies and infertility. Bitches with high titers experienced abnormal estrous cycles. Estradiol rose during proestrus, but instead of falling sharply in early estrus as in controls, it remained elevated. Progesterone did not rise. The moderate-titered bitches had normal cycles and steroid patterns. Bitches immunized with pPZP had moderate titers. Cycles were normal after 3 injections, but after 6 injections one bitch had an abnormal cycle. One pPZP-immunized bitch remained fertile but the others were infertile. Alum was the mildest adjuvant, causing no injection site lesions, but the highest titers occurred with Freund's and CP-20,961 adjuvants. All three adjuvants induced titers sufficient to inhibit fertility. Infertility in bitches immunized with PZP may be due to prevention of zona penetration, because their antisera inhibited zona penetration of oocytes by spermatozoa in vitro. However, alterations in ovarian function preventing ovulation and luteinization could be involved in high-titered bitches.
An important question in mammalian gamete physiology concerns how capacitation and the occurrence of acrosome reactions in motile sperm relate to fertility. Evaluation of these relationships has been restricted by practical limitations because rapid, quantitative assays are unavailable. We have developed a rapid, reproducible assay for the evaluation of acrosomal status utilizing monoclonal antibodies specific to antigens localized in the acrosomal cap region of the sperm head. Mice were immunized with human ejaculated sperm preparations and the resultant hybridomas producing antisperm antibody were selected by solid-phase radioimmunoassay and indirect immunofluorescence (IIF). Two monoclonal antibodies (HS-19, HS-21) recognized target antigens restricted to the acrosomal cap by IIF, and 87 +/- 8.5% of the sperm in fresh ejaculates from 10 different sperm donors showed positive cap fluorescence with these reagents. Loss of HS-21 binding as measured by IIF was correlated with disappearance of the acrosomal cap as observed directly by transmission electron microscopy. Acrosomal disappearance, artificially induced in vitro using the calcium ionophore A23187, also resulted in a loss of HS-21 binding. The induction of acrosomal loss by ionophore was dependent upon extracellular calcium. The data presented suggest that specific monoclonal antibodies can be used for the rapid evaluation of acrosomal status in mammalian sperm.
Seasonal changes in pulsatile luteinizing hormone (LH) secretion in ovariectomized ewes were examined over the course of 2 yr in relation to annual changes in environmental photoperiod, shifts in response to estradiol negative feedback control of LH secretion, and timing of the breeding season. Under natural environmental conditions, the frequency of LH pulses in individual ovariectomized ewes changed gradually and in close association with the annual cycle of day length. As days became shorter in late summer and autumn, LH pulse frequency increased; conversely, as day length increased in late winter and spring, frequency declined. Under artificial conditions in which ovariectomized ewes were exposed to different photoperiods, a similar inverse relationship was observed between day length and LH pulse frequency. The seasonal changes in frequency of LH pulses in ovariectomized ewes, although symmetric with the annual photoperiodic cycle, were not temporally coupled to the dramatic shifts in response to estradiol feedback inhibition of LH secretion at the transitions between breeding season and anestrus. The feedback shifts occurred abruptly and at times when LH pulse frequency in ovariectomized ewes was at, or near, the annual maximum or minimum. The tight coupling between LH pulse frequency and photoperiod leads to the conclusion that there is a photoperiodic drive to the LH pulse-generating system of the ewe. The temporal dissociation between changes in this photoperiodic drive and the seasonal shifts in response to estradiol negative feedback support the hypothesis that the neuroendocrine basis for these two phenomena is not one and the same.