Cycle frequency, length, and vaginal cytology were measured longitudinally in three cohorts of singly housed virgin mice staggered across a 3-year interval. The age profiles of these parameters were qualitatively similar, but quantitatively different, among cohorts. Cycle frequency was initially low (Phase I), due to prolonged cycles and late-starting cycles, and did not peak (Phase II) until mice were 3-5 months old. Phase II lasted for 7-10 months, depending on the cohort. Thereafter cycle frequency declined steadily (Phase III). The average age of cessation of cyclicity varied among cohorts, occurring between 13 and 16 months of age. Age changes in cycle length paralleled those of cycle frequency. During Phase II, median cycle length was less than 5 days and variance was lowest. During Phases I and III, variance was about twofold greater and median cycle length was greater than 5 days. Although median cycle length remained stable for several months during Phase II, the peak period of 4-day cycles was much shorter. In all cohorts, 4-day cycles did not peak until 7-8 months of age and began to decline by 9 months. The decrease in 4-day cycles was associated with a progressive lengthening of cycles-first from 4 to 5 days, then to longer cycles. The fraction of cycles with extended cornification (greater than 2 days) increased with advancing age from less than 0.35 during the initial period of cycle lengthening to a maximum of 0.60. The observation that the initial phase o cycle prolongation was not usually associated with extended cornification is consistent with earlier evidence that this period is characterized by a delayed, rather than prolonged, preovulatory rise of estradiol. However, the increased fraction of prolonged cycles with extended cornification at later ages suggests that the preovulatory elevation of estradiol may ultimately be prolonged.
A repeatable procedure for fertilization of bovine ova in vitro is described. Oocytes were recovered from ovarian follicles or from oviducts near the time of ovulation following treatment of donors with pregnant mare's serum gonadotropin (PMSG) and prostaglandin F2 alpha (PGF2 alpha). For in vitro capacitation semen was incubated, then high ionic strength treated and subsequently incubated in defined medium prior to insemination of oocytes. In one experiment frozen bull semen was successfully used. In experiments with 4 bulls (B, C, D, F), 34 (43.6%) of 78 ova and 13 (19.7%) of 66 follicular oocytes were fertilized in vitro. In the last series (spermatozoa from Bull F) the fertilization of 22 (62.9%) of 35 tubal ova was achieved. In vitro development proceeded to the 8-cell stage. No fertilization in vitro followed use of one male (Bull E), even though his spermatozoa could penetrate zona-free hamster ova in vitro, and higher than usual bacterial contamination of his semen was implicated as the probable cause. Findings suggested vigorous progressive sperm motility and acrosome integrity to be important features of good sperm samples. In one experiment a 4-cell stage embryo was transferred with the result that the recipient gave birth to a normal bull calf on June 9, 1981. The first calf resulting from in vitro fertilization has been found to be completely normal.
Rabbit spermatozoa released from the cauda epididymidis into Tris phosphate medium containing KCl or NaCl and 0.4 mM EDTA underwent spontaneous lipid peroxidation during aerobic incubation at 37 degrees C. In the medium containing 130 mM K+ and O mM Na+ (KTP), the rate of lipid peroxidation, as measured by malonaldehyde production, proceeded at a linear rate of 0.045 nmol malonaldehyde/h per 10(8) cells for 22 h. The motility of these spermatozoa declined with time in medium KTP, with 40% initial forward motility decreasing to zero in 4 h and initial 60% flagellar beating ceasing after 12 h. The percent inert spermatozoa showing no flagellar motion in KTP increased linearly with production of malonaldehyde; all flagellar activity stopped at 0.5 nmol malonaldehyde/10(8) cells. In the Tris phosphate medium containing 120 mM Na+ and 10 mM K+ (NTP), the percentage of sperm showing forward motility was close to 100% and this declined to 60% after 16 h aerobic incubation. Flagellar beating was not observed. In medium NTP, the rate of lipid peroxidation was 0.0056 nmol malonaldehyde/h per 10(8) cells, eightfold lower than that observed in KTP. The same linear correlation between malonaldehyde production and percent inert sperm was found as for KTP: 0.5 nmol malonaldehyde/10(8) cells also corresponded to cessation of flagellar motion. The dependence of motility maintenance on K+ concentration in Tris phosphate medium containing (Na+ + K+)=130 mM showed maximal maintenance at 10 mM K+, with a decline at 0 mM K+ and steep decline at K+ concentrations greater than 30 mM. This strong dependence of rabbit sperm peroxidation on ionic composition of the medium is suggested to involve perturbation of the equilibrium between O2 .- and its conjugate acid species being the agent of peroxidation.
Motility and protein phosphorylation have been measured under identical experimental conditions in ejaculated dog sperm lysed with low concentrations of Triton X-100 and reactivated with [gamma-32P]ATP. Cyclic AMP stimulates motility and protein phosphorylation while calcium inhibits motility and the overall incorporation of phosphate into endogenous proteins. Analysis of 32P-labeled sperm proteins on 1- and 2-dimensional polyacrylamide gels demonstrates that an enhanced phosphorylation of a defined number of specific proteins is associated with cAMP-stimulated motility. A major axonemal proteins, namely tubulin, has been tentatively identified as a phosphoprotein subject to regulation by cAMP. The phosphorylation of tubulin is almost completely dependent upon cAMP and is not affected by microM calcium. On the other hand, the cAMP-dependent stimulated phosphorylation of the other sperm proteins still occurs, but in most instances at a reduced rate in the presence of calcium. Two high molecular weight (Mr) phosphoproteins (350,000 and 260,000 daltons) whose phosphorylation states are modified by cAMP and calcium also were identified. It is suggested that 1 or both these proteins may be high Mr subunits of dynein. The phosphorylation of 1 of these proteins is stimulated by cAMP, but not affected by calcium; the other is stimulated by cAMP and inhibited by calcium. Three major cAMP-independent phosphoproteins of Mr 98,000, 43,000 and 26,000 have been identified. The phosphorylation of the 98,000 Mr protein is markedly reduced by micromolar calcium and not restored by cAMP. Using anticalmodulin drugs to inhibit motility, we suggest that the inhibitory effects of calcium on flagellar motility may be mediated in part by calmodulin. We conclude that the regulation of flagellar motility in cAMP and calcium includes mechanisms involving the control of the phosphorylation state of sperm proteins, some of which may be axonemal components.
The [35S]methionine-labeled proteins secreted from cultured Sertoli cells were analyzed by two-dimensional gel electrophoresis and fluorography. Major polypeptides which were resolved by this procedure were designated by number and further analyzed. Many of these major polypeptides appeared as a series of spots which corresponded to charge isomers. Two of these polypeptides (5 and 6) were shown to be acidic, glycosylated and to comprise the subunits of a dimeric protein of molecular weight 70,000. Some of the polypeptides (4a and 5a) were shown to be secreted from testicular peritubular cells which contaminated the Sertoli cell cultures. However, many of the polypeptides (1,2,3,4,5,5b and 6) were specifically secreted from the Sertoli cells. The fluorogram of the secreted polypeptides obtained from cultured Sertoli cells from 20- or 60-day-old rats were similar to each other but differed from the pattern of polypeptides which were secreted from cultures of Sertoli cells from 10-day-old rats. Polypeptide 3 was identified by immunoprecipitation as testicular transferrin and the synthesis of polypeptide 3 was stimulated when the Sertoli cells were cultured in the presence of follicle-stimulating hormone (FSH), insulin, testosterone and retinol.
Concentrations of estradiol, progesterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin in serum from 6 bitches bled daily for at least 45 days before the onset of proestrus, during proestrus and estrus were determined by radioimmunoassay. Mean concentrations of estradiol in serum were high early in the sampling period (20 to 46 pg/ml) and appeared to decrease prior to the onset of proestrus (8 to 19 pg/ml). There were sporadic increases in serum concentrations of LH throughout the sampling period in each bitch. Five of the 6 bitches sampled had increased serum concentrations of LH following the low mean concentration of estradiol just before the onset of proestrus. Mean concentrations of FSH were highest during anestrus (240 to 294 ng/ml) and near the time of the preovulatory surge of LH (297 ng/ml) and were lowest during proestrus (131 to 200 ng/ml). The mean concentration of progesterone for the 6 bitches remained at less than 1.0 ng/ml throughout late anestrus, but increased to greater than 1.0 ng/ml the day of the maximum mean concentration of LH (preovulatory LH surge). Mean concentrations of prolactin were variable throughout the sampling period and demonstrated no consistent pattern among bitches. The results of the current investigation suggest that neither the canine ovary nor pituitary are quiescent during anestrus. The bitch appears to have sufficient FSH present during anestrus for follicular growth. Serum concentrations of LH appear to increase prior to the onset of proestrus when concentrations of estradiol are lowest, possibly inducing a new follicular phase. Progesterone and prolactin do not appear to be involved in the termination of anestrus in the bitch.
The mechanism whereby glucocorticoids directly inhibit gonadotropin-stimulated testosterone production was studied by using primary cultures of testicular cells from adult hypophysectomized rats. Testicular cells were maintained in serum-free media with hormone treatments administered on Day 8 and media collected 48 h later for steroid and cAMP measurement. Highly purified human chorionic gonadotropin (hCG) increased testosterone production relative to controls. Concomitant administration of either natural (cortisone greater than deoxycorticosterone = aldosterone) or synthetic (dexamethasone greater than or equal to prednisolone) corticosteroids inhibited hCG-stimulated testosterone production in a dose-dependent manner. Dexamethasone at 10(-7) M decreased testosterone production by approximately 50-60% and this inhibitory effect was reversible upon removal of the glucocorticoid. In the presence or absence of a phosphodiesterase inhibitor, dexamethasone decreased hCG-stimulated cAMP production by approximately 60%. Dexamethasone also decreased testosterone production induced by cholera toxin and (Bu)2 cAMP by 43 and 63%, respectively. The dexamethasone suppression of testosterone production was accompanied by marked decreases in androstenedione (80% decrease) and 17 alpha-hydroxyprogesterone (57%) production, with a lesser effect on progesterone production (28% decrease) and no effect on pregnenolone production. Exogenous progesterone and 17 alpha-hydroxyprogesterone augmented hCG-stimulated testosterone production. Dexamethasone reduced the conversion of exogenous progesterone to testosterone by 33% but did not affect the conversion of 17 alpha-hydroxyprogesterone to androstenedione and testosterone, suggesting a specific inhibition of 17 alpha-hydroxylase. These results suggest that glucocorticoids directly suppress Leydig cell steroidogenesis by decreasing gonadotropin stimulation of cAMP production and the activity of 17 alpha-hydroxylase.
Round spermatids were prepared from rat testes and incubated with various substrates (glucose, fructose, pyruvate, lactate and acetate) to measure utilization of substrates and production of ATP in the presence of saturating levels of each substrate. By both criteria lactate is the preferred substrate by a factor of 3 or 4. Production of more than half of the ATP with lactate is substrate is prevented by addition of an inhibitor of alpha-ketoacid dehydrogenase (5-methoxyindole-2-carboxylic acid) Pyruvate and lactate are interconverted and pyruvate inhibits production of ATP from lactate. Synthesis of ATP with lactate and with pyruvate is inhibited by rotenone, rutamycin or 2,4-dinitrophenol. Utilization of glucose is limited by aldolase activity. These findings suggest that exogenous lactate is oxidized by lactate dehydrogenase followed by pyruvate dehydrogenase and Krebs; cycle enzymes under conditions which do not allow pyruvate to inhibit lactate dehydrogenase. ATP is synthesized through electron transport. Post-mitochondrial supernate from spermatids showed that high concentration of pyruvate (greater than 1 mM) inhibit lactate dehydrogenase with pyruvate as substrate and that with lactate as substrate, pyruvate behaves as a competitive inhibitor of lactate dehydrogenase. Evidently lactate is the preferred substrate for round spermatids and energy production is most efficient when this substance is present in high concentrations and pyruvate is present in low concentrations. Reasons are given for suggesting that Sertoli cells may provide the relatively large amounts of lactate required by round spermatids.
Mature rabbit spermatozoa from the cauda epididymidis suspended in potassium Tris phosphate buffer at 24 degrees C produced O2.-, as measured by reduction of acetylated ferricytochrome c, with an intrinsic rate of 0.20 nmol/min per 10(8) cells. This rate increased to 1.80 nmol/min per 10(8) cells in the presence of 10 mM cyanide. These spermatozoa contain 2.8 units per 10(8) cells of superoxide dismutase activity, 95% of which is sensitive, and 5% of which is insensitive, to cyanide inhibition. These activities correspond to the cytosolic Cu-Zn form and the mitochondrial Mn form of the dismutase, respectively. Only the cyanide-sensitive form is released from the sperm on hypo-osmotic treatment or sonication. Hypo-osmotically treated rabbit epididymal spermatozoa produced O2.- with an intrinsic rate of 0.24 nmol/min per 10(8) cells, which increased to 0.58 nmol/min per 10(8) cells in the presence of 10 mM cyanide. Both intact and hypo-osmotically treated cells react with O2.- in a second order reaction as inferred from the hyperbolic dependence on cell concentration of O2.- production rate in both the absence and presence of cyanide. The second order rate constant for this reaction with intact cells, kS, was calculated to be 22.9 X 10(-8) (cells/ml)-1 min-1 in its absence. For hypo-osmotically treated cells, the values of kS were 10.8 X 10(-8) (cells/ml)-1 min-1 and 8.2 X 10(-8) (cells/ml) -1 min-1, respectively. Since hypo-osmotically treated cells have lost much of their plasma membrane, the lower value of kS for the treated cells implies that this membrane is one site of reaction of O2.- with the cells. The increase in kS in the presence of cyanide, which inhibits superoxide dismutase and so increases O2.- production, suggests that the cells become more reactive with O2.- as its production rate increase, as would be expected for the occurrence of radical chain oxidation. This in turn suggests that superoxide dismutase plays a major role in protecting rabbit sperm against damage from lipid peroxidation.
Metabolism of arachidonic acid and prostaglandin F2 alpha by bovine blastocysts and endometrial slices recovered on Days 16 and 19 postmating was studied in vitro. In Experiment 1, arachidonic acid (10 microCi tritiated and 200 micrograms radioinert) was added to blastocysts and endometrial slices prior to incubation for 24 h. [3H]arachidonic acid ([3H]AA) and metabolites in extracts of culture medium and tissue homogenates were separated on columns of Sephadex LH-20. Elution profiles of [3H]AA and metabolites in extracts of culture medium revealed that 13, 14-dihydro-15-keto-PGF2 alpha (PGFM), Pge2, PGF2 alpha, and at least four unidentified compounds were produced by Day 16 and Day 19 blastocysts. Endometrial slices from both days of pregnancy produced 3H-prostaglandins. Experiment 2 was conducted to quantify PGE2, PGF2 alpha and PGFM in aliquots of culture medium from Day 16 and Day 19 blastocyst and endometrial incubates. These tissues were incubated with 200 micrograms of radioinert arachidonic acid. Day 16 blastocysts produced less (microgram/blastocyst; P less than 0.01) of each prostaglandin than Day 19 blastocysts (PGE2, 0.7 +/- 0.4 vs. 4.2 +/- 1.0; PGF2 alpha, 2.1 +/- 0.7 vs. 22.8 +/- 4.1; PGFM, 0.03 +/- 0.01 vs. 0.5 +/- 0.2). Endometrial slices produced PGE2, PGF2 alpha and PGFM, but quantities were not affected by day postmating or uterine horncorpus luteum relationships. The third experiment was conducted to determine directly if Day 19 blastocysts and endometrial slices metabolized [3H]PGF2 alpha to [3H]PGFM. Blastocysts and endometrial slices produced [3H]PGFM. Endometrial slices metabolized 34.3 +/- 1.5% of the [3H]PGF2 alpha to [3H]PGFM, while blastocysts metabolized 7.5 +/- 1.6% of the [3H]PGF2 alpha to [3H]PGFM. Results of this study indicate that bovine blastocysts and endometrial slices can metabolize [3H]AA in vitro. It is postulated that prostaglandins of blastocyst and endometrial orgin have a role in maintenance of early pregnancy in cattle.