The effect of catecholamines and their antagonists on the fertilization of cumulus-free mouse ova in vitro at a suboptimal spermatozoal density

A suboptimal sperm concentration was used to assess the capacity of catecholamines to stimulate the fertilization of cumulus free F₁,(C57BL × CBA) mouse ova in vitro. At a concentration of 50 μM, (L) epinephrine significantly increased the proportion of ova fertilized at 2 × l0⁵ spermatozoa/ml. However, when (D, L) propranolol at an equimolar concentration was tested for inhibition of the (L) epinephrine effect, fertilization was inhibited in both the test and control dishes. At l0μM, propanolol by itself or in the presence of 50μM (L) epinephrine significantly increased the number of ova fertilized at 2 × l0⁵ sperm/ml. Norepinephrine (50 μM) and phentolamine (50 μM), either alone or together, were also slightly stimulatory. Some data are presented to suggest that propranolol may act in a nonadrenergic manner to precipitate the acrosome reaction and that the stimulatory effect is maximised when it is added to spermatozoa at the same time as ova addition. It was suggested that propranolol may act to trigger calcium influx by a nonspecific alteration in membrane function for example in (Ca + Mg) ATPase activity. It was concluded that spermatozoa at suboptimal densities are capable of achieving fertilization and that sperm concentration dependency in fertilization in vitro may be a reflection of the proportion of spermatozoa achieving capacitation.     

Genetic influences on mouse sperm capacitation in vivo and in vitro

Intial in vivo studies were performed to observe the proportion of eggs fertillized at specific intervals after natural mating and ovulation in our research mouse colony. Proestrous females of the C57BL/10Wt, SJL/Wt inbred strains and the F₁ hybrid cross (B10 × SJL or reciprocals) were paired in the after-noon with males of their respective strain and examined for vaginal plugs at the midpoint of the dark period (2400 hours). Oviducts were periodically collected from mated females, and ovulation was first observed at 4, 5.2, and 3 hours after 2400 hours in the B10, SJL, and F₁ hyrid, respectively. The clutch of eggs from each ovulating female, was placed in culture, and cleavage oviduct lavage verifying female mating was placed in culture, and cleavage was used as the criterion for fertilizaition. Fifty percent of the eggs were fertilized 2.2, 5.0, and 2.5 hours after ovulation in B10, SJL, and F₁ hybrid females, respectively. Because twice the legth of time was required to fertilize a similar proportion of eggs from the SJL strain as the F₁ hybrid, these two strains were used for determining their rate of fertilization under more fully controlled conditions in vitro. Forty-nine percent of F₁ hybrid eggs were fertilized after 4 hours incubation with SJL epididymal sperm, whereas 53% fo SJL and 56% of F₁ hybrid eggs were fertilized after only 2 hours incubation with F₁ hybrid epididymal sperm. Thus, using sperm from these two mouse strains, the amount of time required to fertilize approximately 50% of the eggs within a clutch both in vivo and vitro was very similar. These observations demonstrte teh validity of using this in vitro system for fertilization studies and confirm that the temporal events in sperm capacitation and egg penetration are dependent on the genotype of the sperm. Similarities in fertilization rates at specific times after ovulation or insemination in vitro imply that the initiationof sperm capacitation in vivo occurs near the time of ovulation and several hours after mating. We tentatively suggest that follicular fluid may be required for completion of mouse sperm capacitaiton in vivo.     

Efficacy of TEPA as a “sperm label” for competitive fertilization studies in the rabbit

In this paper, the conditions necessary to use TEPA [tris (1-aziridinyl)] effectively as a label for spermatozoa in competitive fertilization are established. The fertilizing ability of rabbit spermatozoa treated with 0 and 0.8 mg TEPA/ml was compared at insemination doses of 1, 5, 20, and 40 × 10⁶ spermatozoa. Fertility was assessed by collecting ova from 64 does 48 to 52 h after insemination. TEPA blocked all but 4% of the ova from developing when 1 × 10⁶ spermatozoa were inseminated, but fertility was reduced. When 5 × 10⁶ spermatozoa were inseminated following treatment with 0, 0.6 or 1.2 mg of TEPA/ml, the fertility was 83, 74 and 50% (P<0.05), and the percentage of ova containing more than four blastomeres was 83, 11 and 5% (P<0.05), respectively. The 0.6% TEPA level was selected for a competitive fertilization trial. Equal numbers of sperm from pure Dutch-color and albino sires were combined so that either both types were untreated, only the ‘albino’ semen was treated, only the ‘Dutch’ semen was treated, or both were treated. Does were inseminated with 5 × 10⁶ sperm and allowed to kindle. The litter sizes were 5.6, 3.1, 2.7, and 0 young, and the proportion of Dutch-color progeny was 63, 97, 0 and 0%, respectively, confirming the effectiveness of TEPA as a “label”. Only one of 60 young born resulted from fertilization by a TEPA-treated spermatozoon, demonstrating that few embryos fully escape the TEPA block. Thus, the TEPA concentration and sperm numbers were established to use TEPA effectively as a label for spermatozoa in competitive fertilization studies.     

Fertilization by sperm injection in the rabbit

Whole rabbit spermatozoa and isolated sperm nuclei were microinjected directly into the ooplasm of hamster and rabbit ova. These injected sperm decondensed and formed male pronuclei during subsequent in-vitro culture. Injection of whole spermatozoa and sperm nuclei prepared by a protocol known to allow in-vitro capacitation of ejaculated spermatozoa yielded a significantly higher (P < 0.01) number of activated rabbit ova containing male pronuclei than did injection of uncapacitated epididymal sperm nuclei or ejaculated sperm nuclei. Rabbit ova fertilized by sperm injection were capable of undergoing normal-appearing cleavage division during 22 h of culture.     

Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova

Several reports have indicated that sperm capacitation includes loss of membrane cholesterol (Chol) with a concomitant decrease in the Chol-to-phospholipid (PL) ratio. Methods were developed for quantifiable removal of bovine sperm Chol, which predisposed sperm to induction of the acrosome reaction upon addition of lysophosphatidylcholine (LPC). The objective of this study was to evaluate the effect of Chol removal from bovine sperm on penetration of zona-free hamster and intact bovine ova in vitro. Washed ejaculated bovine sperm were incubated (2 h, 39°C) in a modified Tyrode's solution (TALP) containing (1) Chol-free liposomes (—Chol, 50 × 10⁶ sperm and 600 nmol phospholipid/ml); (2) liposomes containing 30 mol% Chol (+ Chol, 2 × 10⁸ sperm and 300 nmol total lipid/ml); or (3) no liposomes (Control). We have previously shown that net Chol efflux from sperm is 31% of the total sperm Chol with —Chol liposomes and less than 1% with control media. Sperm were then washed twice and challenged with LPC bound to bovine serum albumin (BSA) using celite as a carrier. Treated sperm (25 × 10⁶) were incubated immediately with either zona-free hamster ova (HO) or in vitro matured bovine ova (BO) in 50-μl droplets of TALP under medical fluid in an atmosphere of 5% CO₂ in air (3 h, 39°C). Ova were fixed in ethanol:acetic acid, stained with 1% orcein, and examined. Percent penetration (%P) of HO (X ± SEM) for 30 and 40 μg of LPC/mg BSA was 59.4 ± 5.3 and 82.9 ± 5.4; 38.5 ± 5.6 and 52.3 ± 4.7; and 16.0 ± 4.6; and 23.2 ± 5.6 for —Chol, Control, and + Chol treatments, respectively (n = 3). %P of BO (X ± SEM) for 30, 35, and 40 μg of LPC/mg BSA was 43.3 ± 5.4, 70.7 ± 7.5, and 81.5 ± 5.1 for —Chol and 16.4 ± 6.9, 36.2 ± 6.9, and 44.2 ± 8.6 for Control treatments, respectively (n = 3). In a second set of experiments %P of BO (X ± SEM) was 63.6 ± 6.8, 31.8 ± 4.9, and 10.5 ± 3.4 for —Chol, Control, and +Chol treatments, respectively, when 40 μg LPC/mg BSA was added (n = 2). %P and the number of sperm per fertilized ovum were consistently higher for the —Chol treatment for both HO and BO (P < .01). These results demonstrate that Chol removal from bovine sperm facilitates penetration of ova in vitro suggesting a potential role in bovine sperm capacitation.     

Subpopulations of human T lymphocytes. II. Effect of thymopoietin, corticosteroids, and irradiation.

Lymphoid cells from normal SJL/J mice gave high proliferative responses but failed to develop cytotoxic activity to γ-irradiated cells from syngeneic transplantable reticulum cell sarcomas (X-RCS). In spite of a vigorous in vivo proliferative response to X-RCS, cytotoxic activity was never generated to detectable levels in vivo. After repeated injections of X-RCS, spleen and, to a lesser degree, lymph node cells acquired the ability to give moderate secondary cytotoxic responses in vitro upon co-culture with X-RCS. This immunity was T-cell mediated and specific for RCS although it did not distinguish between different transplantable RCS lines. SJL/J mice also developed resistance to RCS growth after injection of X-RCS, which showed a transient RCS-line-specific component. (SJL/J × C₅₇B1/6)F₁ mice showed 60% less RCS growth than did SJL/J mice, and their lymphoid cells gave slightly lower proliferative responses than did cells from SJL/J mice, whereas (SJL/J × BALB/c)F₁ mice showed little tumor growth, and their spleen cells proliferated only minimally to X-RCS. B10.S mice allowed moderate RCS growth. Cytotoxic activity was generated in co-cultures with X-RCS of immunized F₁ spleen cells even after a single immunization in vivo but not in cultures of normal F₁ cells with X-RCS.     

Caffeine promotes in vitro fertilization of mouse ova within 15 minutes

Epididymal sperm were collected from C57Bl6/J X DBA2/J (B6D2) males and allowed to capacitate for 2 hr. When cumulus-free oocytes were exposed to sperm for 15 min in either the presence (6.0 mM) or absence of caffeine, fertilization did not occur. However, when cumulus cells were left intact, 23% of oocytes were fertilized in caffeine-free medium and 62% in caffeine-containing medium. When cumulus-free oocytes were incubated with sperm for 30 min, none was fertilized in the absence of caffeine, but 33% were fertilized when 6.0 mM caffeine was present (P less than .02). These effects of caffeine were on the sperm, as sperm exposed to caffeine and then coincubated with oocytes for 15 min in essentially caffeine-free media fertilized a similar percent of oocytes (93%) as when sperm and oocytes were exposed to caffeine during the fertilization period (86%). When sperm were capacitated in caffeine-containing medium, the percentage of ova fertilized was similar to capacitation without caffeine. We conclude that both cumulus cells and caffeine speed up the fertilization process with mouse gametes and that the effect of caffeine is on the sperm, but not due to more rapid capacitation.     

Capacitation of hamster spermatozoa with the divalent cation chelators D-penicillamine,L-histidine,and L-cysteine in a protein-free culture medium

By using a chemically defined (protein-free) culture medium that supports sperm viability but not capacitation or the acrosome reaction, we have determined that hamster spermatozoa can be chemically capacitated in vitro by the divalent cation chelators D-penicilla-mine, L-histidine, and L-cysteine in the absence of bovine serum albumin (BSA). Washed cauda epididymal spermatozoa were preincubated (1–2 × 10⁶ sperm/ml) for 3, 4, or 6 hr at 37°C in 5% CO₂ in air. The basic culture medium used for sperm preincubation and for sperm:egg coincubation was a modified Tyrode's solution (protein-free) containing 10 mM sodium lactate, 100 μM sodium pyruvate, and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm viability was maintained in all preincubation and coincubation media with PHE (20 μM D-penicillamine, 100 μM hypotaurine, and 1.0 μM epinephrine). The low control sperm preincubation medium consisted of TLP-PVA. In some cases the high control preincubation medium also contained 3 mg/ml BSA (TALP-PVA). The experimental preincubation medium was TLP-PVA with additional D-penicillamine (125 or 500 μM), or L-histidine (10, 100, or 1,000 μM) or L-cysteine (25, 75, or 125 μM). After preincubation, sperm were coincubated (2 × 10⁴ sperm/ml) with cumulus-free hamster eggs in TALP-PVA ± additional D-penicillamine, L-histidine, or L-cysteine for 1.5 hr, fixed, and evaluated for percent egg penetration as an index of sperm capacitation. The results demonstrate that hamster spermatozoa can be chemically capacitated in vitro with D-penicillamine (500 μM: range of mean penetration values, 53.6%–84.3%), L-histidine (100 μM: range of mean values, 24.8%–56.3%) or L-cysteine (75 μM: 51.3%) in the absence of exogenous protein.     

Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl₂ and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl₂ revealed that plasma membrane was removed only from the acrosomal region and remained predominately intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl₂ lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg²⁺, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM-PL) for 2 hours. Under these culture conditions the addition of Ca²⁺ (5 mM) to the capacitated spermatozoa induced approximately 78 ± 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 ± 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg²⁺, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2-deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.     

Promotion of capacitation of guinea pig spermatozoa by the membrane mobility agent,A2C,and inhibition by the disulfide-reducing agent,DTT

The membrane mobility agent A₂C accelerates the onset of the acrosome reaction of guinea pig spermatozoa by promoting capacitation. Spermatozoa incubated in a suspension of A₂C particles in Ca²⁺-free medium for one hour undergo a synchronous, rapid acrosome reaction upon the addition of Ca²⁺. These acrosome-reacted spermatozoa are capable of fertilization as assessed by their ability to penetrate (fuse with) zona-free hamster eggs. The disulfide-reducing agent, dithiothreitol (DTT) inhibits A₂C-mediated capacitation. It also blocks fertilization of zone-free eggs by acrosome-reacted spermatozoa by preventing attachment of the spermatozoa to the egg plasma membrane. The mode of A₂C action on spermatozoa is compared to that of A₂C-induced fusion in somatic cells. The similarity of the molecular events in the sperm membrane during capacitation and the acrosome reaction to these in other fusion events is pointed out. Inhibition of capacitation by DTT points to the importance of membrane and/or submembrane proteins and thiol groups in this process. Oxidation of sperm membrane SH groups may play an important role in in vivo capacitation.     

Sperm volumetric dynamics during in vitro capacitation process in bovine spermatozoa

Previous studies have demonstrated that sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in bovine spermatozoa. In this study, sperm head morphometry was used to investigate its value as a biophysical marker for detecting volumetric changes in bovine spermatozoa under in vitro capacitating and non-capacitating incubation conditions. To further test this hypotesis, aliquots of pooled, washed bovine sperm were incubated in either Tyrode’s complete medium with heparin (TCMH; a capacitating medium containing Ca²⁺, NaHCO₃ and heparin), Tyrode’s complete medium heparin-free (TCM; a medium containing just Ca²⁺ and NaHCO₃) or Tyrode’s basal medium (TBM; a non-capacitating medium free of Ca²⁺, NaHCO₃ and heparin, used as control). Aliquots of sperm were processed for morphometric analysis at different incubation-time intervals (0, 3 and 6 h at 38°C), and the chlortetracycline assay was used simultaneously to confirm the ability of the sperm to undergo capacitation (B pattern) and the acrosome reaction (AR pattern) status in each medium. After 3 h of incubation under TCMH conditions, a significant increase was observed in the percentage of B and AR patterns and a significant decrease was found in all sperm morphometric parameters (P<0.01). Interestingly, after 6 h of incubation in TCMH, the percentage of B and AR patterns increased drastically over time and marked differences were found in the dimensional and shape parameters, which were significantly smaller compared with TBM or TCM media (P<0.001). Significant correlations were observed between sperm size and AR pattern (r=−0.875, P<0.01). In conclusion, sperm head morphometry can be used as a potential biophysical marker for detecting volumetric changes during capacitation process in bovine spermatozoa.     

Estimation and manipulation of glutathione levels in prepuberal mouse ovaries and ova:Relevance to sperm nucleus transformation in the fertilized egg

Glutathione (GSH), the major low-molecular-weight thiol in mammalian cells, is believed to be a necessary factor for the transformation of the disulfide-stabilized sperm nucleus into the male pronucleus after fertilization. Its concentration in mouse ova, isolated from the ampulla of the oviduct after hormone-induced superovulation of 3–4-week-old mice, has been determined by an enzymic cycling microassay. The level found was 1.80 pmol per ovum. Mean ovum diameter was estimated as 71–72 μm, indicating a GSH concentration of 9–10 mM in the mouse egg. Administration of L-buthionine S, R-sulfoximine, an inhibitor of GSH biosynthesis, during the 2 days preceding ovulation, reduced ovum GSH content below 0.20 pmol (<1.0 mM). The mean GSH concentration of the hormone-stimulated ovaries was reduced from 3.2 mM to 0.2 mM under these conditions. It has also been demonstrated that measurement and manipulation of ovum and ovarian levels of GSH can aid in studying its function in ovaries, ova, and early embryos. Hormone-induced superovulation was achieved in BSO-treated prepuberal C57B1/6 X SJL mice whose ovaries contained less than 10% of control levels of GSH. Over 50% of the isolated ova were fertilized in vitro. However, abnormal one-cell embryos resulted in which the maternally derived pronucleus coexisted with an unchanged sperm nucleus, thus confirming that adequate levels of GSH are necessary for initiating transformation of the fertilizing sperm nucleus.     

Incubation of mouse sperm with lactate delays capacitation and hyperactivation and lowers fertilization levels in vitro

Mouse sperm were incubated in medium with or without 24 mM lactate and assessed for (1) motility characteristics including hyperactivation—a computer-assisted motion analysis system was used; (2) capacitation—a chlortetracycline fluorescent dye binding assay was used; and (3) ability to penetrate oocytes. Lactate affected all aspects of motility and delayed the rates of both hyperactivation and capacitation. When a concentration of 8 × 10³ sperm/ml was used for insemination in vitro, sperm preincubated 60–90 minutes in medium with lactate prior to insemination in lactate-free medium fertilized fewer oocytes than did sperm preincubated in lactate-free medium. Use of a calcium-sensitive electrode demonstrated that lactate chelated appreciable amounts of calcium in the medium. Capacitation was assayed in sperm incubated 60 minutes in medium with various concentrations of lactate or CaCl₂. When medium containing lactate was compared to medium without lactate but having a similar level of free calcium, the level of capacitation of sperm incubated with lactate was less than half that of sperm incubated without lactate. These results demonstrate that including 24 mM lactate in the medium can have detrimental effects on mouse sperm hyperactivation and capacitation. The detrimental effects on capacitation are partly but not completely due to the chelation of calcium by lactate.     

Fertilization of cumulus-free,zona-intact mouse ova in vitro at high and low sperm concentrations

The effect of varying the sperm concentration between 2 × 10⁵ sperm/ml and 8 × 10⁶ sperm/ml on fertilization of cumulus-free, zona-intact F1 (CBA × C57BL) mouse ova by QS and F1 (CBA × C57BL) mouse spermatozoa was studied. The spermatozoa from both strains of mice exhibited optimal fertilization rates at 2 × 106 sperm/ml. However, at sperm concentrations greater than 4 × 106 sperm/ml and less than 1 × 106 sperm/ml, fertilization rates were significantly reduced. F1 spermatozoa were more susceptible to dilution than QS spermatozoa. A significant interaction between strain and sperm concentration indicated that the two strains produced different fertilization rates at different sperm densities. Extracts of epididymal fluid, medium from capacitated spermatozoa, or ampulla fluid did not improve the fertilization rate at 2 × 105 sperm/ml, but retaining the cumulus oophorus did. The decrease in fertilization rate at 8 × 106 sperm/ml can in part be attributed to a nondialysable inhibitor from the neat sperm preparation that appeared to be of epididymal origin.     

Embryo production in superovulated Angus cows inseminated four times with sexed-sorted or conventional, frozen-thawed semen

This study tested the hypothesis that four inseminations of commercially frozen sexed semen (≥2.1 × 10⁶ sperm per 0.25-mL straw) in superstimulated embryo donors would yield a percentage and quantity of transferable embryos similar to that achieved with conventional frozen semen. Bos taurus, angus cows (n = 32), stratified by age and body condition, were randomly allocated to receive four inseminations of frozen-thawed semen, either conventional semen (≥15 × 10⁶ sperm/straw; Conventional) or sexed semen (≥2.1 × 10⁶ sperm/straw; Sexed) from one of two AI sires. From 10 to 13 d after estrus, follicle-stimulating hormone (FSH) was given twice-daily, with prostaglandin F_2α given twice on the last day. Cows were inseminated once (1×) at first detected estrus and twice (2×) and once (1×) at 12 and 24 h later, respectively, with nonsurgical embryo recovery 7 d after first detected estrus. The study was repeated 30 d later (switch-back experimental design). The total number of ova per flush was similar between Conventional and Sexed treatments (10.9 ± 1.8 vs. 10.5 ± 1.6), but the number of Grade 1 embryos was greater (P < 0.01) for Conventional (4.3 ± 0.8 vs. 2.3 ± 0.7). Conversely, the mean number of unfertilized ova was greater (P < 0.05) for Sexed (5.6 ± 1.0 vs. 3.0 ± 1.2). There was no significant difference between treatments for numbers of degenerate, Grades 2 or 3, and transferable embryos and no significant differences between bulls in percentage of transferable embryos (44.4% and 46.7%). However, fertilization rates and percentage of transferable embryos were affected (P < 0.05) by period and donor. In conclusion, superstimulated donor cows inseminated four times had fewer Grade 1 embryos and more unfertilized ova with sexed versus conventional semen.     

Capacitation of bovine spermatozoa by lysophospholipids and trypsin

Bovine spermatozoa were incubated in vitro with lysophosphatidylserine (LPS), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), or trypsin. Capacitation of sperm was evaluated by penetration of the zonae pellucidae of dead bovine oocytes. Capacitation times could be shortened to 3 h or less by treatment of spermatozoa with each of these lysophospholipids (LPLs) (P < .05). The maximum oocyte penetration percentages for individual LPLs were 40% for 10 μM LPS, 24% for 160 μM LPC, 31% for 320 μM LPE, and 19% for 320 μM LPI. Capacitation also was facilitated (P < .01) by trypsin treatment of spermatozoa. Spermatozoa treated with 250 or 2,500 units/ml of trypsin penetrated more oocytes (17 and 18%) than spermatozoa treated with 0 or 25 units/ml of trypsin (0 and 3%). Spermatozoa treated with increasing concentrations of LPL showed a decrease in both the percentage of intact acrosomes and of progressively motile spermatozoa. Increasing levels of trypsin in the incubation medium also led to a decrease (P < .05) in the percentages of intact acrosomes and a decrease (P < .01) in the percentages of progressively motile spermatozoa. Percentages of live, ovulated oocytes fertilized by spermatozoa incubated for 1 h in LPS (86%, 6/7) were not different from those incubated for 24 h in control medium (71%, 5/7). Percentages of oocytes fertilized with both of these capacitation treatments were higher (P < .05) than for oocytes exposed or killed or uncapacitated sperm. Rapid induction of capacitation and the acrosome reaction can be accomplished by exogenous treatment of bovine sperm with lysophospholipids or trypsin.     

PTH release stimulated by high extracellular potassium is associated with a decrease in cytosolic calcium in bovine parathyroid cells

We employed the calcium (Ca⁺⁺)-sensitive, intracellular dye QUIN-2 to examine the role of cytosolic Ca⁺⁺ in the stimulation of PTH release by high extracellular potassium (K⁺) concentrations. Addition of 55 mM KCl to cells incubated with 115 mM NaCl and 5 mM KCl lowered cytosolic Ca⁺⁺ at either low (0.5 mM) extracellular Ca⁺⁺ (from 194±14 to 159±9 nM, p<.01, N=6) or high (1.5 mM) extracellular calcium (from 465±38 to 293±20 nM, p<.01, N=10). This reduction in cytosolic Ca⁺⁺ was due to high K⁺$\text{per}\text{se}$ and not to changes in tonicity since addition of 55 mM NaCl was without effect while a similar decrease in cytosolic Ca⁺⁺ occurred when cells were resuspended in 60 mM NaCl and 60 mM KCl. PTH release was significantly (p<.01) greater at 0.5 and 1.5 mM Ca⁺⁺ in QUIN-2-loaded cells incubated with 60 mM NaCl and 60 mM KCl than in those exposed to 115 mM NaCl and 5 mM KCl. In contrast to most secretory cells, therefore, stimulation of PTH release by high K⁺ is associated with a decrease rather than an increase in cytosolic Ca⁺⁺.     

In vitro fertilizing characteristics of bovine spermatozoa with multiple nuclear vacuoles: A case study

A study was designed to determine the in vitro fertilizing characteristics of bovine semen with a high percentage of spermatozoa with multiple nuclear vacuoles. In Experiment 1, a total of 620 oocytes was divided into 2 groups and inseminated with spermatozoa from 1 of 2 different bulls at a concentration of 2 × 10⁵/ml. After Percoll washes, 73.5 ± 3.0% of spermatozoa from Bull A contained multiple nuclear vacuoles, while no sperm cells from Bull B contained vacuoles. After 19.5 ± 0.5 h of co-incubation of oocytes with spermatozoa, loosely attached sperm cells were removed by washing, and the oocytes were fixed between 2 poly-l-lysine coated glass slides. Mean (±SD) percentage of fertilization was significantly lower (P < 0.05) in Bull A (19.7 ± 7.0%) than in Bull B (67.6 ± 4.5%). In one-third of the oocytes fertilized by spermatozoa from Bull A, sperm head decondensation was incomplete and normal male pronucleus formation did not occur. All oocytes fertilized by Bull B had normally decondensed sperm heads. Although fewer (P < 0.05) spermatozoa from Bull A were bound to the zona pellucida than from Bull B, the percentage of vacuolated sperm cells bound to the zona pellucida (73.3 ± 7.8%) did not differ from that in the inseminate. The mean number of sperm cells binding to fertilized oocytes was higher than to unfertilized oocytes for both bulls (P < 0.05). In Experiment 2, 748 salt-stored oocytes (zonae) were inseminated with semen from the same 2 bulls to determine the ability of spermatozoa to penetrate the zona pellucida. The percentage of zonae penetrated by spermatozoa from Bull A (69.9 ± 3.5%; a mean of 2.4 ± 2.3 spermatozoa) was lower (P < 0.05) than from Bull B (96.5 ± 14.7%; a mean of 11.3 ± 9.9). Although the proportion of vacuolated sperm cells from Bull A that bound to the zona pellucida did not differ from that in the inseminate, the proportion of those penetrating the zona pellucida (52.7%) was lower (P < 0.05). In summary, vacuolated sperm cells apparently gained access to the oocyte and bound to the zona pellucida, but they penetrated the zona pellucida at a lower rate and apparently did not form normal male pronuclei.     

Studies on Ergothioneine. IX. Ergothioneine Stimulates Mouse Spermatozoa and Fertilization in vitro

The effects of ergothioneine on spermatozoa and ova were investigated in vitro and in vivo. Spermatozoa were treated with ergothioneine in vitro , and injected into the uterine cavity of female mice immediately after the induction of superovulation. The ova were recovered 24 hr later and assessed for fertilization. Preincubation of spermatozoa with ergothioneine resulted in a significant increase in the fertilization rate. When ova were inseminated in the same manner in vitro with spermatozoa treated with 0.1 or 1.0 mM of ergothioneine, the penetration rate was significantly increased. These results suggest that ergothioneine is effective in inducing both capacitation and the acrosome reaction of mouse spermatozoa. Ergothioneine at concentrations of 0.1 and 1.0 mM in the preincubation medium was also effective in inducing the acrosome reaction of guinea pig spermatozoa. However, it had no significant effect on the development of 2-cell ova in vitro .     

Association of chronotype and social jetlag with human non-verbal intelligence

The aim of this study was to evaluate the effects of chronotype and social jetlag (SJL) on intelligence. Subjects were aged 14–25 years (n = 1008). A significant effect of intelligence on academic performance, as measured by the Raven’s Standard Progressive Matrices test, was found (F_2,917 = 11.75, P < 0.0001, η² = 0.03). When SJL was less than 2 hours, the intelligence of people with late chronotype was found to be higher than that of subjects with early and intermediate chronotypes (F_2,305 = 3.12, P < 0.05, η² = 0.02). A negative effect of SJL on the results of intelligence testing was noted only in subjects with late chronotype (F_2,536 = 2.61, P < 0.05, η² = 0.02). Our data suggest that people with late chronotype have a higher level of intelligence, but these advantages disappear when SJL ≥2 hours.