The proportion of living sperm in semen from six representative mammals was assessed by means of a dual staining technique using the stains SYBR-14 and propidium iodide (PI). SYBR-14, a newly developed fluorescent nucleic acid stain, maximally absorbs at 488 nm and emits at 518 nm when bound to DNA. Microscopic examination revealed that SYBR-14 stained the nuclei of living sperm bright green as determined by simultaneous examination of fluorescence and motility. Conversely, PI stained only nonmotile sperm that had lost their membrane integrity. Sperm from bulls, boars, rams, rabbits, mice, and men were stained and examined through use of fluorescence microscopy. The proportions of living and dead sperm were determined by first staining with SYBR-14 and PI and then assessing stain uptake by flow cytometry. Similar staining patterns were observed in all six mammalian species tested. Three populations of sperm were identified: livingSYBR-14 stained, deadPI stained, and moribunddoubly stained. The SYBR-14 staining was replaced by PI staining as sperm progressed from living to moribund. The transition from green (SYBR-14) to red (PI) fluorescence started at the posterior region of the sperm head and proceeded anteriorly. The proportions of living and dead sperm in mammalian semen were readily identified through use of dual staining with SYBR-14 and PI and quantified through use of flow cytometry.
Mechanisms regulating responses of the ovine uterus to endocrine and paracrine signals during the estrous cycle and pregnancy are likely to require tissue- and cell-specific regulation of steroid hormone receptor gene expression. To determine effects of day and pregnancy status (cyclic or pregnant) on uterine estrogen receptor (ER) and progesterone receptor (PR) gene expression, ewes were hysterectomized either on Day 1 (Day 0 = estrus/mating), 6, 11, 13, or 15 of the estrous cycle (n = 3/day) or on Day 11, 13, 15, 17, or 25 of early pregnancy (n = 5/day). Steady state levels of ER and PR mRNA were determined in endometrial and myometrial tissues by slot-blot hybridization and ribonuclease protection assays, respectively, using homologous ovine ER and PR cRNA probes. Changes in spatial expression of ER and PR mRNA and protein in uterine tissue sections were determined by in situ hybridization and immunocytochemical analyses. In cyclic ewes, steady state levels of endometrial ER mRNA were highest on Day 1, declined between Days 1 and 6, and increased between Days 11 and 15. However in pregnant ewes, endometrial ER mRNA levels decreased between Days 11 and 15 and increased slightly between Days 15 and 25. In cyclic ewes, levels of myometrial ER mRNA were highest on Day 1, decreased to Day 6, and remained low thereafter. In cyclic ewes, endometrial PR mRNA levels were highest on Day 1, decreased between Days 1 and 11, and then increased between Days 13 and 15. In cyclic ewes, myometrial PR mRNA levels were highest on Day 1 and declined thereafter. Endometrial PR mRNA levels were not different between cyclic and pregnant ewes on Days 11, 13, and 15. In pregnant ewes, PR mRNA levels were low on Day 11, increased between Days 11 and 17, and decreased between Days 17 and 25. In pregnant ewes, myometrial PR mRNA levels were low and did not change between Days 11 and 25. In situ hybridization and immunocytochemical analyses revealed distinct tissue- and cell type-specific alterations in uterine ER and PR mRNA and protein expression during the estrous cycle and early pregnancy that generally paralleled overall changes in steady state levels of ER and PR mRNAs. In the endometrium, the most striking observation was that PR mRNA and protein expression disappeared from the luminal and shallow glandular epithelium between Days 6 and 13 of the estrous cycle, whereas ER mRNA and protein expression was low on Days 6 and 11 and increased between Days 11 and 15 in the luminal and shallow glandular epithelium. During early pregnancy, expression of ER and PR mRNAs, as well as ER and PR protein, was very low or absent in the luminal and shallow glandular epithelium between Days 13 and 25 of pregnancy. Moreover, ER and PR mRNA and protein were consistently present at low levels in the stroma and deep glandular epithelium in both cyclic (Days 11-15) and pregnant (Days 11-25) ewes. Collectively, results suggest that uterine ER and PR gene expression is regulated in a tissue- and cell type-specific manner during the estrous cycle and early pregnancy.
Several investigators have demonstrated that LHRH agonists (LHRHa) inhibit ovarian follicular depletion induced by chemotherapeutic agents in rodents. It is not clear whether or not the same effects occur in primates. Six adult female rhesus monkeys underwent unilateral ovariectomy and were divided into two groups that received monthly injections of either Lupron depot (LHRHa) or placebo vehicle. Both groups received cyclophosphamide (CTX) injections. Weekly blood samples were assayed for FSH, estradiol and progesterone. Mean serum FSH levels significantly increased in the CTX group and significantly decreased in the LHRHa+CTX group. At the end of treatment, the remaining ovary was removed and serially sectioned, and ovarian follicle number and size were analyzed. CTX resulted in a significant reduction of nonprimordial follicles < 50 microns in diameter. The rate of loss of primordial follicles was expressed as a percentage of the original follicle count. During the treatment period, 64.6 +/- 2.8% of the total primordial follicles were lost in the CTX group compared to only 28.9 +/- 9.1% in the LHRHa+CTX group (p < 0.05). The percentage rate of decline per day was 0.120 +/- 0.012 for the CTX group compared to 0.057 +/- 0.019 (p < 0.05) for the LHRHa+CTX group. The findings indicate that LHRHa can protect the ovary against CTX-induced damage in rhesus monkeys.
The objectives of the present experiment were to compare survival after transfer of bovine embryos produced in vivo with those produced in vitro and to examine the physical characteristics of fetuses produced from these transfers. Embryos produced in vivo (Holstein x Angus) were recovered from uterine flushings of superovulated heifers 7 days after first artificial insemination, and embryos produced in vitro (Holstein x beef breeds) were collected 7 days after insemination. Embryos were paired by source (in vivo, in vitro), stage (compact morula, blastocyst), and quality grade (excellent = 1, good = 2), and transferred nonsurgically to recipient heifers on Day 7 (+/- 1 day) of the estrous cycle. Pregnancy status was monitored by determination of serum progesterone concentrations, ultrasonography, and palpation through 7 mo of gestation, at which time fetuses were recovered. In comparison with grade 1 embryos produced in vivo, the risk of embryonic death after transfer was similar for grade 2 embryos produced in vivo (p = 0.56) and for grade 1 embryos produced in vitro (p = 0.88). By contrast, grade 2 embryos produced in vitro were at greater (p = 0.04) risk of embryonic death. Embryo loss was associated (p = 0.01) with increased serum concentrations of progesterone in recipients at the time of transfer. At 7 mo of gestation, fetuses from embryos produced in vitro were heavier (p = 0.02) than fetuses from embryos produced in vivo and had skeletal measurements that were disproportionate (p < or = 0.04) to body weight.
The Leydig cell of the testis is the only cell in the male that has the capacity to synthesize testosterone from cholesterol. Testosterone is critical during fetal development for male sexual differentiation, and postnatally for initiation and maintenance of spermatogenesis and the expression of the male secondary sex characteristics. The biosynthesis of testosterone requires the activities of four enzymes, cholesterol side-chain cleavage enzyme (P450scc), 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD), 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha)), and 17-ketosteroid reductase. The expression of these enzymes appears to be regulated by different mechanisms. The recent isolation of the mouse cDNAs and structural genes that encode these enzymes has enabled us to begin to investigate the regulation of their expression at the molecular level. This review discusses the regulation by cAMP and steroids of three enzymes in Leydig cells, P450scc, P450(17 alpha), and 3 beta HSD, as well as characterization of the promoters of the mouse genes that encode P450scc and P450(17 alpha).
This study shows that the nucleus of the secondary spermatocyte can participate in syngamy and normal embryonic development. Spermatogenic cells were released from the seminiferous tubules of adult mice, and the secondary spermatocytes were selected according to the size of the whole cell and nucleus. The accuracy of this selection, evaluated by chromosome analysis, was 86%. Nuclei of presumptive secondary spermatocytes were freed from the cytoplasm and then injected individually into mature oocytes. This process itself did not activate the oocytes. The oocytes were electroactivated about 2 h after injection, at which time prematurely condensed chromosomes of the spermatocyte had become associated with the microtubules of a spindle. Following activation, the chromosomes of both the oocyte and spermatocyte completed their second meiotic division, culminating in the extrusion of two separate polar bodies and the formation of one male and one female pronucleus in about 75% of the oocytes into which spermatocyte nuclei had been injected. Two- or four-cell embryos arising from such oocytes were randomly selected and transplanted to foster mothers. Twenty-four percent developed into normal fertile offspring. The young born later to these offspring were all normal. The results of this study indicate that gametic imprinting of mouse spermatogenic cells is completed either in the testis before the second meiotic division or within the cytoplasm of a mature oocyte after artificial nuclear transfer.
It is well established that the epididymis is the site where spermatozoa are matured and stored, but our understanding of the regulation of epididymal epithelium functions and their effects on spermatozoa is still fairly limited. The most active regulator of epididymal functions seems to be dihydrotestosterone, the 5 alpha-reduced metabolite of testosterone. Our laboratory has focused on the regulation of 5 alpha-reductase, with studies encompassing its messenger RNA, protein and enzyme activity. We have also investigated the hormonal regulation and distribution of other specific key proteins found in epididymal epithelial cells that play critical roles in the function of these cells. These proteins include clusterin or sulfated glycoprotein-2 and the glutathione S-transferases (GST). Using complementary experimental approaches, including orchidectomy and hormonal replacement, efferent duct ligation, and developmental studies, we have established that 5 alpha-reductase enzyme activity is present in both nuclear and microsomal fractions; the nuclear enzyme appears almost exclusively in the initial segment of the epididymis. In addition, 5 alpha-reductase activity and the mRNAs for both the type 1 and type 2 form of the enzyme are regulated differentially with respect to age and site within the epididymis. Immunolocalization of the protein has revealed that it is located in principal cells and that its subcellular location is dependent on the region of the epididymis. These results indicate that there is both transcriptional and post-transcriptional regulation of the expression of 5 alpha-reductase. Clusterin is a hydrophobic protein secreted by Sertoli cells and found in high concentration in the epididymis. This glycoprotein is expressed at its highest levels in the initial segment and caput epididymidis and at very low levels in the corpus and cauda epididymidis of the intact rat, and it exhibits a novel pattern of androgen regulation. In the areas of highest expression, there is no androgen dependence; however, orchidectomy causes a dramatic increase in the message for clusterin, which is suppressible by androgens in the segments where expression is normally lowest. The GSTs are a family of enzymes thought to play a key role in detoxification. Members of the GST family are expressed in a region-dependent manner along the rat epididymis. We have found that the localization of one member of this enzyme family, GST P, or subunit Yp, is selective for basal cells in the corpus and cauda epididymidis.
Osmotic permeability characteristics and the effects of cryoprotectants are important determinants of recovery and function of spermatozoa after cryopreservation. The primary purpose of this study was to determine the osmotic permeability parameters of human spermatozoa in the presence of cryoprotectants. A series of experiments was done to: 1) validate the use of an electronic particle counter for determining both static and kinetic changes in sperm cell volume; 2) determine the permeability of the cells to various cryoprotectants; and 3) test the hypothesis that human sperm water permeability is affected by the presence of cryoprotectant solutes. The isosmotic volume of human sperm was 28.2 +/- 0.2 microns3 (mean +/- SEM), 29.0 +/- 0.3 microns3, and 28.2 +/- 0.4 microns3 at 22, 11, and 0 degrees C, respectively, measured at 285 mOsm/kg via an electronic particle counter. The osmotically inactive fraction of human sperm was determined from Boyle van't Hoff (BVH) plots of samples exposed to four different osmolalities (900, 600, 285, and 145 mOsm/kg). Over this range, cells behaved as linear osmometers with osmotically inactive cell percentages at 22, 11, and 0 degrees C of 50 +/- 1%, 41 +/- 2%, and 52 +/- 3%, respectively. Permeability of human sperm to water was determined from the kinetics of volume change in a hyposmotic solution (145 mOsm/kg) at the three experimental temperatures. The hydraulic conductivity (Lp) was 1.84 +/- 0.06 microns.min-1.atm-1, 1.45 +/- 0.04 microns.min-1.atm-1, and 1.14 +/- 0.07 microns.min-1.atm-1 at 22, 11, and 0 degrees C, respectively, yielding an Arrhenius activation energy (Ea) of 3.48 kcal/mol. These biophysical characteristics of human spermatozoa are consistent with findings in previous reports, validating the use of an electronic particle counter for determining osmotic permeability parameters of human sperm. This validated system was then used to investigate the permeability of human sperm to four different cryoprotectant solutes, i.e., glycerol (Gly), dimethylsulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG), and their effects on water permeability. A preloaded, osmotically equilibrated cell suspension was returned to an isosmotic medium while cell volume was measured over time. A Kedem-Katchalsky model was used to determine the permeability of the cells to each solute and the resulting water permeability. The permeabilities of human sperm at 22 degrees C to Gly, DMSO, PG, and EG were 2.07 +/- 0.13 x 10(-3) cm/min, 0.80 +/- 0.02 x 10(-3) cm/min, 2.3 +/- 0.1 x 10(-3) cm/min, and 7.94 +/- 0.67 x 10(-3) cm/min, respectively. The resulting Lp values at 22 degrees C were reduced to 0.77 +/- 0.08 micron.min-1.atm-1, 0.84 +/- 0.07 micron.min-1.atm-1, 1.23 +/- 0.09 microns.min-1.atm-1, and 0.74 +/- 0.06 micron.min-1.atm-1, respectively. These data support the hypothesis that low-molecular-weight, nonionic cryoprotectant solutes affect (decrease) human sperm water permeability.
Naturally occurring antiphospholipid antibodies are strongly associated with placental dysfunction and severe obstetrical complications. We have produced three monoclonal antiphospholipid antibodies that differentiate between phosphatidylserine (PS)- and cardiolipin (CL)-dependent antigens, 3SB9b (CL-/PS+), BA3B5C4 (CL+/PS+), and D11A4 (CL+/PS-). We tested these monoclonal antiphospholipid antibodies in an assay for intertrophoblastic fusion. A JAR choriocarcinoma cell line was induced to undergo intercellular fusion by forskolin in the presence or absence of monoclonal antiphospholipid antibodies. The amount of syncytium formation was quantified by using fluorescein isothiocyanate (FITC)-conjugated anti-desmosome antibody to visualize intercellular membranes and propidium iodide to stain nuclei and by counting those cells with multiple nuclei. Without the presence of antiphospholipid antibodies, and in cultures containing BA3B5C4 (CL+/PS+) or D11A4 (CL+/PS-), approximately 70% of JAR formed syncytial cells after 24 h of forskolin treatment. Less than 13% of the cells formed synctia in 2-day cultures that were not exposed to forskolin or that contained forskolin in the presence of 3SB9b (CL-/PS+). These data suggest that phosphatidylserine is externalized during intertrophoblastic fusion and that antiphospholipid antibody with reactivity against PS, but not CL, can affect placental development by interfering with the normal formation of syncytiotrophoblast.
The objective of this study was to examine the expression and activation of the c-kit receptor, a specific receptor for kit ligand (stem cell factor, steel factor), in rat type A spermatogonia. Testes were obtained from 9-day-old rats, decapsulated, and then subjected to sequential enzymatic digestion. The mixture of testicular cell types was then separated by sedimentation velocity at unit gravity. The isolated type A spermatogonia were characterized by light and electron microscopy. They exhibited spherical nuclei containing several nucleoli and associated chromatin clumps and organelles generally in a perinuclear location similar to that found in the in vivo 9-day-old testis. The synthesis of the c-kit receptor by the spermatogonia was established by hybridization of total RNA with a specific cDNA for mouse c-kit receptor. Two mRNA transcripts migrating at 4.8 kb and 12 kb were observed. Localization of the c-kit receptor in the isolated cells was determined by immunocytochemistry using an antibody to c-kit protein. Specific staining for c-kit receptor was observed in the cytoplasm of the isolated type A spermatogonia. Furthermore, the presence of the c-kit receptor protein in the spermatogonia was confirmed by Western blot analysis using the same antibody. The antibody recognized the c-kit receptor at approximately 160 kDa. In an attempt to determine whether this receptor has a functional significance, we examined the effect of kit ligand on the phosphorylation of the c-kit receptor. The c-kit receptor appeared to be constitutively autophosphorylated on tyrosine at low basal levels, and upon stimulation with kit ligand, the amount of phosphorylated protein increased significantly. These observations indicate that kit ligand induces autophosphorylation of the c-kit receptor, which may lead to the activation of other cellular target proteins responsible for spermatogonial proliferation and/or differentiation.