Showing papers in "Biology of Reproduction in 1991"

Effect of stress on the activity of the hypothalamic-pituitary-gonadal axis: peripheral and central mechanisms.

Abstract: This article reviews the mechanisms believed to mediate stress-induced inhibition of reproductive functions and the anatomical sites at which these effects take place. Particular emphasis is placed on the potential modulating role of hormones or neurotransmitters released during stress. At the level of the gonads, adrenal corticoids, pro-opiomelanocortin (POMC)-like peptides, and corticotropin-releasing factor (CRF) are reported to interfere with the stimulatory action of gonadotropins on sex steroid-producing cells. Increased circulating corticosteroid levels may also decrease pituitary responsiveness to GnRH. There is, however, increasing evidence that these mechanisms are primarily involved in mediating the effects of prolonged stress, but not those of an acute stimulus. In contrast, a variety of hormones or neurotransmitters, including CRF, POMC peptides, and biogenic amines act within the brain to mediate the inhibitory influence of both acute and prolonged stresses on reproductive function.

DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells.

Abstract: Mammalian sperm DNA is the most tightly compacted eukaryotic DNA, being at least sixfold more highly condensed than the DNA in mitotic chromosomes. To achieve this high degree of packaging, sperm DNA interacts with protamines to form linear, side-by-side arrays of chromatin. This differs markedly from the bulkier DNA packaging of somatic cell nuclei and mitotic chromosomes, in which the DNA is coiled around histone octamers to form nucleosomes. The overall organization of mammalian sperm DNA, however, resembles that of somatic cells in that both the linear arrays of sperm chromatin and the 30-nm solenoid filaments of somatic cell chromatin are organized into loop domains attached at their bases to a nuclear matrix. In addition to the sperm nuclear matrix, sperm nuclei contain a unique structure termed the sperm nuclear annulus to which the entire complement of DNA appears to be anchored when the nuclear matrix is disrupted during decondensation. In somatic cells, proper function of DNA is dependent upon the structural organization of the DNA by the nuclear matrix, and the structural organization of sperm DNA is likely to be just as vital to the proper functioning of the spermatozoa.

A major human epididymis-specific cDNA encodes a protein with sequence homology to extracellular proteinase inhibitors.

Abstract: The amino acid sequence of a major human epididymis-specific protein was deduced from the nucleotide sequence of its cloned cDNA. The encoded product showed characteristics of a secretory protein, with a signal peptide followed by a small (approximately 10-kDa), acidic (pI 4.3), and cysteine-rich polypeptide. The positions of half-cysteines suggested that it was a two-domain member of the family of 'four-disulfide core' proteins to which a number of proteinase inhibitors belong. Southern blot analyses of human genomic DNA showed that the transcripts originated from a single copy gene. Northern blot and in situ transcript hybridization specifically localized the HE4 (human epididymis gene product) mRNA to the epithelial cells of the epididymal duct, predominantly within the distal sections. A possible function in sperm maturation as indicated by amino acid similarities to extracellular proteinase inhibitors of genital tract mucous secretions is discussed in the context of its tissue-specific transcription.

Hormonal regulation of uterine secretion of prostaglandin F2 alpha during luteolysis in ruminants.

Abstract: In recent years, considerable progress has been made in our understanding of the endocrine mechanisms that control the pattern and timing of uterine secretion of prostaglandin F2 alpha (PGF2 alpha) during luteolysis in ruminants. Oxytocin may be important in establishing a pulsatile pattern of secretion. Neurohypophyseal oxytocin appears to be released in a pulsatile fashion and may initiate each episode of PGF2 alpha secretion from the uterus. Uterine PGF2 alpha stimulates release of oxytocin from the corpus luteum. Luteal oxytocin further stimulates secretion of PGF2 alpha from the uterus and may induce a transient refractoriness of the uterus to subsequent stimulation with oxytocin. Uterine refractoriness subsides after approximately 6 h. A similar desensitization phenomenon occurs in response to PGF2 alpha at the level of the corpus luteum. Together, uterine and luteal refractoriness may account for the interval between pulses of PGF2 alpha observed during luteolysis. Uterine secretory responsiveness to oxytocin increases at luteolysis, when endogenous, pulsatile secretion of PGF2 alpha normally begins. Thus, the acquisition by the uterus of responsiveness to oxytocin may determine when endogenous secretion of PGF2 alpha occurs during the estrous cycle. Uterine secretory responsiveness to oxytocin develops slowly, in the presence of progesterone. Progesterone exerts two types of effects that contribute to the regulation of PGF2 alpha secretion. First, prolonged exposure to progesterone appears to promote uterine accumulation of arachidonic acid, prostaglandin endoperoxide synthase, and other substances needed for synthesis of PGF2 alpha. Second, progesterone exerts a suppressive effect on secretion, which wanes after prolonged exposure. Together, these effects of progesterone ensure that PGF2 alpha is secreted only at the appropriate time to induce luteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Fertilizing capacity of bovine sperm may be maintained by binding of oviductal epithelial cells.

Abstract: The ability of the bovine oviduct to maintain the motility and fertilizing capacity of bovine sperm was investigated by incubating frozen-thawed sperm with endosalpingeal epithelial cells cultured on either tissue culture plastic (nonpolarizing) or Matrigel.coated Millicell (polarizing) substrata, Sperm were also incubated in medium alone or with cultured bovine tracheal epithelial cells, Motility was determined at 6-h intervals over a 48-h period. The fertilizing capacity of sperm was evaluated after 0, 24, and 30 h of incubation by adding oocytes to the culture and determining the incidences of fertilization and polyspermy. Motility was maintained for 48 h in sperm that bound to endosalpingeal epithelial cells, but to a greater extent with polarized cells (38.4% motile) than with nonpolarized cells (0.8%). Fertilizing capacity was maintained for 30 h in sperm incubated with endosalpingeal epithelial cells on Matrigel/Millicell, but not in sperm incubated in medium alone or with tracheal cells. Only sperm incubated with oviductal cells developed hyperactivated motility. Scanning electron micrographs revealed that sperm were bound by the rostral portion of the intact acrosome to the apical surface of polarized endosalpingeal cells, These results suggest that the oviduct may not only store sperm but may also maintain sperm viability and fertilizing capacity during the preovulatory period,

Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II.

Abstract: Two gonadotropins, GTH I and GTH II, were isolated from pituitaries of spawning coho salmon (Oncorhynchus kisutch) using sequential extractions with ammonium acetate (pH 9.0) and 40% ethanol, precipitation with 80% ethanol, gel filtration chromatography (Sephadex G-100), anion-exchange chromatography (Mono-Q Sepharose), and gel filtration chromatography (Sephadex G-75). Coho salmon GTH I and GTH II stimulated steroidogenesis in vitro in a similar dose-dependent manner when incubated with either ovaries or testes of prepubertal coho salmon. An in vivo bioassay using coho salmon parr demonstrated that coho salmon GTH I and GTH II did not contain thyrotropic activity. Molecular weights were estimated by gel filtration chromatography to be 43,000 and 39,000 for GTH I and GTH II, respectively. Analysis of coho salmon GTH I and GTH II on reversed-phase high-performance liquid chromatography (rpHPLC) revealed that they consist of alpha and beta subunits with N-terminal amino acid residues of Tyr, Gly (alpha, beta of GTH I) and Tyr,Ser (alpha, beta of GTH II). Coho salmon GTH I-beta and GTH II-beta differed from each other in amino acid composition, N-terminal amino acids (Gly vs. Ser), and molecular weights in SDS-PAGE (19,000 vs. 20,000) and had a high degree of chemical similarity to chum salmon GTH I-beta and GTH II-beta, respectively. Specific rabbit antisera to the beta subunits of coho salmon GTH I and GTH II were generated. The observation of two GTHs with distinctly different chemical characteristics in coho salmon is similar to what has previously been found in chum salmon.

In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, protein-free culture media.

Abstract: Development of bovine embryos derived from in vitro-matured/in vitro-fertilized (IVM/IVF) oocytes was examined in two culture media: hamster embryo culture medium (HECM), a relatively simple, chemically defined, protein-free medium containing 20 amino acids; and tissue culture medium (TCM)-199, a more complex medium designed for culture of somatic cells. The first experiment studied (1) effects of glucose and/or phosphate (Pi) using HECM and (2) the development of bovine IVM/IVF embryos in four different conditions: HECM, TCM-199, TCM-199 + 10% unheated bovine calf serum (BCS), and oviduct cell-conditioned TCM-199 + 10% BCS. After IVF, 45% of the inseminated oocytes developed to the morula/blastocyst stages (MB blastocyst development was depressed in the presence of glucose and Pi when compared to Pi alone. When the four culture conditions were compared, there was no significant difference in M&B development (42-51% of inseminated oocytes). However, blastocyst development in TCM-199 supplemented with 10% BCS (29.7%) or with BCS + oviduct cell-conditioned medium (21.6%) was significantly greater than in nonsupplemented HECM (9.7%) or TCM-199 (13.8%). In the second experiment, the effects of serum supplementation and/or oviduct cell conditioning on HECM and TCM-199 were examined in a 2 x 2 x 2 factorial experiment. Oviduct cell conditioning of either HECM or TCM-199 without serum supplementation did not enhance bovine embryo development. Serum supplementation exhibited a biphasic effect, with inhibition at the first cleavage and stimulation of morula compaction and blastocyst formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the function of hyperactivated motility in sperm.

Abstract: After insemination, mammalian sperm undergo a striking change in flagellar beat pattern, termed hyperactivation. In low-viscosity culture medium, nonhyperactivated sperm flagella generate relatively symmetrical, low-amplitude waves, while hyperactivated sperm flagella generate an asymetrical beating pattern that results in nonprogressive movement. Since sperm encounter highly viscous and viscoelastic fluids in the female reproductive tract, the progress of hyperactivated sperm was compared with that of nonhyperactivated and transitional sperm in media of increasing viscosity. Hamster sperm obtained from the caudal epididymis were incubated in a medium that promotes capacitation. After 0, 3, and 4 h of incubation, the majority of the sperm exhibited, respectively, activated, transitional, and hyperactivated motility. At each of these time points, aliquots of sperm were removed from incubation and added to solutions of 0, 5%, 10%, 20%, and 30% Ficoll in medium. Samples containing mostly hyperactivated sperm (4 h) maintained higher swimming and flagellar velocities and were able to generate greater forces in response to increased viscous loading than activated sperm (0 h). Transitional sperm (3 h) showed an intermediate response. The paths of hyperactivated sperm through solutions of 20% and 30% Ficoll were considerably straighter than those made through medium alone. This is the first demonstration that hyperactivation can confer a mechanical advantage upon sperm in the oviduct where they may encounter viscous oviductal fluid and a viscoelastic cumulus matrix.

Morphological and functional heterogeneity in the rat prostatic gland.

Abstract: Ductal morphogenesis and adult ductal branching patterns were examined in the rat prostate by a microdissection method. The rat prostate consists of paired (right and left) subdivisions which correspond in large part to the classically defined lobes: ventral prostate, lateral prostate, dorsal prostate, and coagulating gland. Of particular interest was the finding that the lateral prostate consists of two different ductal zones: (1) lateral type 1 prostate with 5-7 long main ducts (resembling miniature palm trees) that extend cranially towards both the seminal vesicle and dorsal prostate to arborize near the bladder neck, and (2) lateral type 2 prostate with 5-6 short main ducts that arborize caudal to the bladder neck and give rise to compact bushy glands. Both lateral prostatic groups had a ductal-acinar organization. The adult structure of the other rat prostatic lobes was also examined, and closely resembled their mouse counterparts. The ventral prostate, which had 2-3 pairs of slender main ducts per side, and the coagulating gland, which had 1 main duct per side, was completely ductal in structure. In contrast, the dorsal prostate, which had 5-6 pairs of main ducts per side, had a ductal-acinar structure. Ductal branching morphogenesis occurred at different rates in different lobes and was essentially complete in the prostate at the 30 days. Immunocytochemical studies with an antibody to DP-1, a major secretory protein of the rat dorsal prostate, revealed that secretory function was initiated at approximately 30 days after birth in the coagulating gland, the dorsal prostate, and lateral type 1 prostate. A consistent feature of the lateral type 2 prostate was the absence of DP-1. On Western blots, DP-1 was detected in the secretion of the coagulating gland, lateral type 1 and dorsal prostate, but not in the ventral and lateral type 2 prostate. Polyacrylamide gel electrophoresis confirmed this result and demonstrated that the lateral type 2 prostate expressed several low-molecular weight secretory proteins not found in the other lobes of the prostate. On the whole, the rat prostate exhibited considerable heterogeneity both between and within lobes in developmental processes, ductal patterning, histology, and functional expression.

The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones.

Abstract: The objective of this study was to investigate the possible effect of recombinant bovine somatotropin (BST) on ovarian folliculogenesis and ovulation rate. Twelve Hereford X Friesian heifers received daily injections of either 25 mg BST (6 heifers) or vehicle (6 heifers) for a period of two estrous cycles until slaughter. Blood samples were collected three times a week for measurements of peripheral growth hormone (GH), insulin-like growth factor I (IGF-l), FSH, LII, estradiol, and progesterone. Serial blood samples were also taken every 10 min for 8 h on Days 12 and 19 of the second estrous cycle to monitor GH, IGFI, FSH, and LH profiles. At the end of treatment (Day 7 of the third estrous cycle), the heifers were killed and their ovaries were collected. Ovulation rate was determined by counting the number of fresh corpora lutea (CL). All antral follicles�2 mm in diameter were dissected to assess antral follicle populations. Granulosa and thecal cells from the three largest follicles and CL from each heifer were collected for FSH and LII binding measurements. All heifers had a single ovulation. The treated heifers had significantly more antral follicles (60.2 ± 6.7) than did the animals in the control group (33.2 ± 3.2) (p 10 mm, 5-10 mm, and 2-5 mm in diameter were 0.8 ± 0.2, 6.8 ± 1.4, and 52.5 ± 6.5 for the treated group, and 0.8 ± 0.2, 6.5 ± 1.0, and 25.8 ± 2.7 for controls. The treated group had significantly higher peripheral concentrations of GH and IGF-l throughout the treatment period (p 0.05). FSH and LH concentrations throughout the treatment period and LII pulse frequency and pulse amplitude during the mid-luteal and follicularphases of the estrous cycle also showed no significant differences between the two groups (p > 0.05). There was no effect of treatment on FSH or LII binding to granulosa and thecal cells or on LII binding to CL In conclusion, BST treatment increased the population of antral fofficles2-5 mm in diameter in mature heifers. This effect

Quantitative cell composition of human and bovine corpora lutea from various reproductive states.

Abstract: The cell composition of human and bovine corpora lutea (CL) from various reproductive states was investigated by computerized video-based interactive Bioquant image analysis system IV and by light microscope immunocytochemistry. Human and bovine CL contained more nonluteal cells than luteal cells. Human CL contained a lower number of luteal and a greater number of nonluteal cells than bovine CL. Regardless of the reproductive state, human CL contained more small luteal cells than large luteal cells. In all reproductive states except in the late luteal phase, the bovine CL also contained more small luteal cells than large luteal cells. The average sizes of all the cells in human CL were smaller than in bovine CL. Human CL contained more vascular space than bovine CL during mid and late luteal phases. The number of luteal cells increased and nonluteal cells decreased from early to mid luteal phase, and then luteal cells decreased and nonluteal cells increased in late luteal phase and in degenerating human and bovine CL. While the change of number of small and large luteal cells first occurred from early to mid luteal phase in human CL, it did not take place until the late luteal phase in bovine CL. The average size of large luteal cells in humans and of small luteal cells in cattle did not change, whereas size of the other cells changed in different reproductive states in both human and bovine CL. The cell composition of term pregnancy human CL was similar to mid or late luteal phase, whereas the cell composition of early pregnancy bovine CL was similar to mid luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle.

Abstract: Three experiments were conducted to examine effects of Buserelin, a potent agonist of gonadotropin-releasing hormone, on characteristics of ovarian follicles in cycling cows and heifers. In experiment 1, heifers were injected once with 10 micrograms Buserelin on Day 11, 12, or 13 of the estrous cycle (estrus = Day 0), or once with 20 micrograms of Buserelin on Day 12. Additionally, two groups were injected with a luteolytic dose of prostaglandin F2 alpha (PGF2 alpha) on Day 13 preceded with or without a Buserelin injection (10 micrograms) on Day 12. A control group did not receive a Buserelin injection. Ovaries were recovered and weighed after animals were slaughtered on Day 15. Follicle diameters were measured with calipers. Follicles for all experiments were classified as small (class 1: 3-5 mm diameter), medium (class 2: 6-9 mm), or large (class 3: greater than 9 mm). Heifers receiving only Buserelin had an increased number of medium-sized follicles compared to controls. Buserelin injection administered 24 h before PGF2 alpha reduced the decline in the average weight of the ovaries containing the corpus luteum (7.8 g for Buserelin before PGF2 alpha vs. 6.7 g for no Buserelin before PGF2 alpha). Buserelin pretreatment appeared to delay or prevent complete luteolysis by the injected PGF2 alpha. In experiment 2, 0, or 10 micrograms Buserelin was injected on Day 12 and follicle development was monitored by ultrasonography in situ from Day 12 to estrus. Follicles also were classified as clear or cloudy; cloudy was associated with flocculent material in the follicular fluid or with an indistinct follicular wall.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a bovine pregnancy-associated glycoprotein.

Abstract: A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942).

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

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

Attachment of boar sperm to mucosal explants of oviduct in vitro: possible role in formation of a sperm reservoir.

Abstract: Ejaculated boar sperm were incubated with explants of porcine oviductal mucosa that had been dissected from the isthmic and ampullar regions of gilts. Sperm bound within minutes to the epithelial surfaces of the explants. Binding was not affected by region (isthmus or ampulla) nor day of estrous cycle (Day 0 or Day 10), but was increased by addition of 70 pg/ml 17 beta-estradiol to the medium. Scanning electron micrographs indicated that sperm bound, via the acrosomal region, to ciliated cells. After 24 h, the numbers of bound sperm dropped significantly, but the motility of the bound sperm did not. A mucous material that entrapped sperm was observed on the epithelial surfaces of 23/32 isthmic and only 4/32 ampullar explants. These results indicate that sperm sticking to ciliated cells and mucus can create a sperm reservoir in the isthmus, but the means by which sperm are released remain unknown.

Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat.

Abstract: Sulfated glycoprotein-2 (SGP-2) is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. SGP-2 mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications. The detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. SGP-2 was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins.

Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa cells.

Abstract: Previously described models for avian ovarian steroidogenesis, using mature, 25-40-mm preovulatory follicles as the source of tissues, were based on the assumption that interaction of the granulosa layer, as the predominant source of progesterone, with adjacent theca cells is required for maximal production of C21, C19, and C18 steroids. In the present study, we evaluated the steroidogenic capacity of ovarian cells isolated from less mature, 6-8-mm and 9-12-mm follicles in the chicken ovary (representative of a stage of development 2-3 wk prior to ovulation) to determine at which stage of follicular development granulosa and/or theca cells become steroidogenically competent. Granulosa cells collected from 6-8-mm follicles were found to be virtually incompetent to produce steroids, containing extremely low basal levels of progesterone (12 pg/5 x 10(5) cells) and failing to respond with increased steroid output following a 3-h exposure to ovine LH (oLH; 0.1 and 100 ng/0.5 ml), ovine FSH (oFSH; 100, 500, and 1,000 ng/0.5 ml), 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; 0.33 and 3.33 mM) or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, addition of pregnenolone (20 and 200 ng/0.5 ml) to granulosa incubations resulted in significantly increased progesterone levels. Granulosa cells of 6-8-mm follicles also failed to increase cAMP formation in the presence of oLH (10, 100, and 1,000 ng/0.5 ml) and 3-isobutyl-1-methylxanthine (IBMX; 10 microM), but responded to stimulation with 1,000 ng oFSH (4.4-fold increase over basal) or 10 microM forskolin (32-fold increase over basal) in the presence of IBMX. In contrast, granulosa cells isolated from 9-12-mm follicles and incubated for 3 h in vitro were found to contain basal progesterone levels 200-fold higher than those found in granulosa cells of 6-8-mm follicles. Furthermore, granulosa cells of 9-12-mm follicles markedly increased progesterone production following incubation in the presence of oFSH (100-1,000 ng/0.5 ml), 8-bromo-cAMP (0.33 and 3.33 mM), or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, these granulosa cells remained unresponsive to oLH (0.1, 10, and 100 ng/0.5 ml), failing to increase cAMP accumulation (in the presence of IBMX) and progesterone output. Theca cells of small yellow follicles were found to produce measurable basal levels of progesterone, androstenedione, and estradiol, and levels of each steroid were significantly increased following a 3-h challenge with oLH, 8-bromo-cAMP, 25-hydroxycholesterol, and pregnenolone.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrogen peroxide is involved in hamster sperm capacitation in vitro.

Abstract: We have investigated the possibility that the generation of hydrogen peroxide (H2O2) by spermatozoa plays a physiological role during capacitation. Capacitation is defined as the incubation period required for fertilization in mammals. Capacitation culminates in an exocytotic event, the acrosome reaction (AR). Mammalian sperm generate H2O2 during aerobic incubation and do not contain catalase, the enzyme that promotes scavenging of H2O2. In the present work we show that added catalase inhibited the AR, while glucose oxidase (GO), an enzyme that generates H2O2, accelerated the onset of the AR. Direct addition of H2O2 also stimulated the AR; catalase inhibited both the stimulation by GO and by H2O2. The onset of the AR was always preceded by the appearance of hyperactivated motility. The stimulation of the AR by H2O2 was manifest 1-2 h after the addition of H2O2. Catalase added at 3 h of incubation was less effective in inhibiting the AR than catalase added at the beginning. Incubation of sperm with catalase prevented the induction of the AR by the membrane-perturbing lipid, lysophosphatidyl choline. Taken together, these results suggest that H2O2 produced by hamster sperm plays a significant role during capacitation, possibly in membrane reorganization to facilitate the fusion that takes place during exocytosis of the acrosomal contents.

The energetic regulation of ovulation: a realistic role for body fat.

Abstract: This review weighs the evidence for and against the hypothesis that ovulation is regulated by a critical amount of body fat. The evidence supporting this hypothesis is correlative, and most of it stems from observations made in humans. On balance, the evidence from human studies does not support the hypothesis, however, and the results of animal studies argue strongly against it. In the latter regard, a variety of experimental approaches have been tried in both adult and peripubertal females of several species, and the results almost uniformly show little relationship between fatness and ovulation. There is no doubt that ovulation can be regulated somehow in relation to whole-body energy balance and that fat stores are an important component of energy balance, but there is no reason to accord body fat a direct causal role in regulating ovulation.

Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos.

Abstract: The present study characterized the profile of nuclear remodeling in nuclear transplant rabbit embryos and investigated the relationship between chromatin behavior after transfer and embryo development. The developmental potential and pattern of remodeling of donor nuclei from cleavage-, morula-, and blastocyst- (inner cell mass ICM, and trophectoderm, TE) stage donors were evaluated. In addition, we determined whether a modification in the synchrony between blastomere fusion and oocyte activation altered the profile of nuclear remodeling and affected development of reconstituted embryos. Development to blastocysts was similar with 8- and 32-cell-stage donor nuclei (42% and 33%, respectively, p greater than 0.1). However, it was reduced with ICM transplants (17%, p less than 0.05), and development of TE transplants did not progress beyond the 8-cell stage. Upon blastomere fusion into nonactivated oocyte cytoplasm, nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by pronuclear (PN) formation and swelling. PCC occurred synchronously within 1.2-1.5 h post-fusion with all stages of donor nuclei (p greater than 0.1). PN formation in 8- and 32-cell transplants occurred approximately 4 h after fusion, and was synchronous to that of female pronuclei in activated oocytes; however, it was delayed in ICM and TE transplants (p less than 0.01). With all stages of donor nuclei, final nuclear diameter was similar to, or larger than, that of female pronuclei. Fusion to activated oocyte cytoplasm, as opposed to nonactivated cytoplasm, prevented PCC and extensive nuclear swelling (16.0 +/- 0.7 vs. 30 +/- 0.7 microns, respectively, p less than 0.01). Nuclear diameter in early embryos was smaller (p less than 0.01), and development to blastocysts was reduced (p less than 0.05). The results indicate that remodeling of the donor nucleus is not essential for development to blastocysts; however, it is beneficial. Furthermore, complete reprogramming seems possible only after remodeling of the donor nucleus, i.e., PCC in nonactivated cytoplasm, followed by nuclear swelling upon activation of the oocyte.

The expression of c-kit protein during oogenesis and early embryonic development.

Abstract: The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and was shown to be allelic with the white-spotting locus (W) of the mouse. Mutations at the W locus have pleiotropic effects on the development of hematopoietic stem cells, melanoblasts, and primordial germ cells. In order to elucidate the role of c-kit protein in gametogenesis and oocyte maturation, we have examined immunohistochemically the expression of c-kit in the ovaries of mice at late fetal and postnatal stages, and in early embryos. By the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody, the c-kit protein was detected in ovaries after the time of birth, but not before. The expression of c-kit was observed mainly on the surface of oocytes, but not in granulosa cells nor in interstitial regions. Oocytes of primordial to fully grown Graafian follicles showed the c-kit protein. When ovulation was induced by hCG, the expression of c-kit in ovulated unfertilized oocytes was weaker than in oocytes of Graafian follicles. In 1-cell embryos the c-kit protein was still observed, but with cell division its expression further decreased, and it was not detected in embryos of 4-cell, 8-cell, and morula stages. In summary, the highest expression of c-kit was observed on the surface of oocytes arrested in the diplotene stage of meiotic prophase. With ovulation and the resumption of meiotic maturation, its expression declined. These results suggest that the c-kit protein may play some role in meiotic arrest, oocyte growth, and oocyte maturation.

The effects of soluble products of activated lymphocytes and macrophages on blastocyst implantation events in vitro.

Abstract: The purpose of this study was to investigate the effects of soluble products of activated lymphocytes and macrophages on mouse blastocyst attachment and trophoblast outgrowth in vitro. Hatched blastocysts were incubated with medium alone, supernatant fluids from mixed lymphocyte cultures (MLC), and with individual human and murine lymphokines and monokines in fibronectin-coated wells. Cultures were assessed at 24, 48, and 72 h for blastocyst attachment and trophoblast outgrowth. Both human and murine MLC supernatant fluids significantly enhanced trophoblast outgrowth in vitro. The cytokine, interleukin-1 beta (Il-1 beta), at a concentration of 10(3) U/ml, inhibited blastocyst attachment but significantly enhanced trophoblast outgrowth of attached blastocysts. Granulocyte, macrophage-colony-stimulating factor (GM-CSF) at a concentration of 250 U/ml significantly inhibited blastocyst attachment, while gamma interferon (gamma-IFN) at a concentration of 2.5 x 10(3) U/ml significantly inhibited trophoblast outgrowth and caused degenerative morphological changes in these cells. The results of this study indicate that products of activated immune cells may either facilitate or impede implantation events depending on the types of predominant cytokines present, their concentration(s), and the timing of their secretion relative to embryonic development.

The transition from maternal to zygotic control of development occurs during the 4-cell stage in the domestic pig, Sus scrofa: quantitative and qualitative aspects of protein synthesis.

Abstract: A study was conducted to identify the embryonic stage when the zygotic genome begins to direct development and to characterize protein synthesis in pig oocytes and embryos. Reproductive tracts of gilts were flushed to obtain unfertilized oocytes (UFO), zygotes (Z), 2-, 4-, and 8-cell embryos, compact morulae (M), initial blastocysts (IB), blastocysts (B), and hatched blastocysts (HB). Pig eggs and embryos were cultured in medium containing 1 microM L-[35S]methionine and evaluated for amino acid uptake, incorporation of the radiolabel into protein, and qualitative changes in protein profiles specific to each cleavage stage. Unfertilized oocytes sequestered 65.7 fmol methionine/4 h/embryo. Uptake of methionine decreased (p less than 0.05) from the Z (49.4), 2-cell (41.8), and 4-cell (37.6) embryonic stages to the M (8.97 fmol/4 h/embryo) stage. This downward trend was reversed at the IB, B, and HB stages when uptake increased to 37.3, 50.3, and 84.2 fmol/4 h/embryo, respectively. Incorporation of methionine into protein followed a similar pattern, being relatively higher in the UFO (21.0), Z (20.5), and 2-cell stages (16.0); decreased (p less than 0.05) at the 4-cell (6.67), 8-cell (6.84), and M (6.16) stages; and increased (p less than 0.05) at the IB (28.0), B (41.5), and HB (69.6 fmol/4 h/embryo) stages. Differences in protein profiles were observed for UFO, Z, 4-cell, and M stages using lysates of single embryos, one-dimensional SDS-PAGE, and fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the Presence of a Major Gene Influencing Ovulation Rate on the X Chromosome of Sheep

Abstract: In a flock of highly prolific Romney ewes obtained from industry flocks, one ewe (A281), with a production record of 33 lambs born in 11 lambings, produced a number of female descendants with high ovulation rates The mode of inheritance of this trait was determined in a series of four progeny tests of male descendants of this ewe The first progeny test produced strong evidence for a new major gene affecting ovulation rate in this family line; this finding was supported by two subsequent progeny tests The fourth progeny test was designed to test the hypothesis that this gene is carried on the X chromosome The results showed that six sons of a carrier ram did not inherit the gene, but it was passed on to three of his five maternal grandsons This finding, together with evidence of genetic segregation in the progeny of carrier females, demonstrates for the first time the presence of a major gene for prolificacy specifically located on the X chromosome The effect of the gene is to increase ovulation rate by about one additional egg per ewe

Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro.

Abstract: In previous reports from this laboratory, we identified the presence of a novel alpha-D-mannosidase on the surface of rat, mouse, hamster, and human spermatozoa [J Cell Biol 1989; 109:1257-1267 and Biol Reprod 1990; 42:843-858]. Since it has been suggested that mannosyl residues on the egg zona pellucida may be important for sperm-egg binding, studies were undertaken to examine the potential role of the sperm alpha-D-mannosidase during fertilization. Incubation of mouse spermatozoa in the presence of increasing concentrations of the inhibitory sugars, alpha-methyl mannoside, alpha-methyl glucoside, D-mannose, or D-mannitol, resulted in a dose-dependent decrease in the number of spermatozoa bound per egg without a deleterious effect on sperm motility or on the sperm acrosome, and a dose-dependent inhibition of the sperm mannosidase activity. Galactose, however had no effect on sperm-egg binding or on sperm mannosidase activity. Two nucleotide sugars (UDP-GlcNAc and UDP-gal) were also tested and shown to reduce sperm-egg binding but with only a minimal effect on sperm mannosidase activity. In additional studies, spermatozoa incubated in the presence of a mannose-containing oligosaccharide exhibited a dramatic reduction in sperm-egg binding that correlated with a similar inhibition of sperm mannosidase activity. The oligosaccharide substrate did not affect sperm motility or the sperm acrosome. These studies suggest that the sperm alpha-D-mannosidase may play an important role during fertilization.

Prostaglandin E2 hastens oviductal transport of equine embryos.

Abstract: The hypothesis that treatment of pregnant mares with prostaglandin E2 (PGE2) hastens the oviductal transport of equine embryos was tested by treating bred mares with PGE2 on Day 3 after ovulation and subsequently measuring the rate of hastened oviductal transport (estimated by the uterine embryo recovery rate on Day 4 after ovulation). In a preliminary, noncontrolled experiment, oviductal transport was apparently not hastened after intramuscular, intrauterine, or intraperitoneal PGE2 administration to bred mares (0/6, 0/3, and 0/3 mares, respectively). Oviductal transport appeared to be hastened in 1/13 mares after a single intraoviductal administration of PGE2, and in 2/2 mares after continuous intraoviductal administration of PGE2. In a subsequent, controlled experiment, treatment with a continuous intraoviductal infusion of PGE2 hastened oviductal transport in significantly more (p less than 0.01) mares versus a continuous intraoviductal infusion of vehicle or no treatment (6/11 vs. 0/11 or 0/11 mares, respectively). Unfertilized oocytes and oviductal masses were also recovered from mare uteri after continuous intraoviductal PGE2 administration, but were not recovered after vehicle administration or no treatment. These results support the hypothesis that PGE2 treatment hastens the oviductal transport of equine embryos, and suggest a role for embryonic PGE2 in the initiation of selective oviductal transport in the mare.

Role of epithelial cells of the male excurrent duct system of the rat in the endocytosis or secretion of sulfated glycoprotein-2 (clusterin).

Abstract: The localization of sulfated glycoprotein-2 (clusterin; SGP-2) was investigated in the rete testis, efferent ducts, and epididymis of the rat using light (LM) and electron (EM) microscope immunocytochemistry. At the LM level, the epithelial cells of the rete testis and efferent ducts demonstrated an intense immunoperoxidase reaction over their apical and supranuclear regions, and sperm in the lumen of the efferent ducts were unreactive. In the EM, gold particles were found exclusively over the endocytic apparatus of these cells. In the proximal area of the epididymal initial segment, an insignificant immunostaining of epithelial cells and sperm was observed. However, the distal area of the initial segment showed a moderate staining over the epithelial principal cells and sperm, while in the intermediate zone of the epididymis a stronger reaction was observed over these cells. The strongest immunoperoxidase reaction was noted in the caput epididymidis, where it formed a distinct mottled pattern. Thus, while some principal cells were intensely stained, others were moderately or weakly stained; a few were completely unreactive. In the corpus and cauda epididymidis, the staining pattern was similar but not as intense. In the EM, only the secretory apparatus of these cells was found to be immunolabeled with gold particles. Sperm in the lumen of these different regions were also labeled. The epithelial clear cells were unreactive throughout the epididymis. Northern blot analysis substantiated these results and showed the presence of highest levels of SGP-2 mRNA in the caput epididymidis, especially in its proximal area, whereas increasingly lower levels were found in the corpus and cauda epididymidis. In summary, these results suggest that testicular SGP-2 dissociates from the sperm during passage through the rete testis and efferent ducts, where it is endocytosed by the epithelial cells lining these regions. In the epididymis, it is replaced by an epididymal SGP-2 that is secreted by the epithelial principal cells of the epididymis. Furthermore, in the epididymis, the principal cells appear to be in different functional states with respect to the secretion of epididymal SGP-2 within a given region of the duct as well as along the epididymal duct.

Diet-induced hyperlipidemia in cattle modifies the intrafollicular cholesterol environment, modulates ovarian follicular dynamics, and hastens the onset of postpartum luteal activity.

Abstract: Three experiments were conducted with female cattle during the postpartum period and during the estrous cycle to examine the effects of diet-induced hyperlipidemia on lipoprotein-cholesterol and triglyceride (TG) metabolism, ovarian follicular dynamics, and incidence of postpartum luteal activity. Dietary-lipid effects were examined independently of metabolizable energy intake. Feeding a high-lipid (HL) diet (8% total lipid) for the first 3 wk of the puerperium (Experiment 1) increased (p less than 0.0001) the concentration of total cholesterol and high-density lipoprotein-cholesterol (HDL-CH), but not TG, within follicular fluid (FF) of estrogen-active (E-A) and inactive (E-I) follicles. Increases (1.4- to 1.7-fold) were similar in proportion to those observed in peripheral blood serum, but absolute concentrations were about 45% of that in serum. Greater than 95% of the cholesterol in FF was HDL-CH, with a greater (p less than 0.06) proportion of HDL-CH sequestered by E-A compared to E-I follicles. The HL diet increased (p less than 0.06) the number of medium-sized (3.1-9.9 mm) follicles present at ovariectomy 19-21 days postcalving and increased (p less than 0.03) concentrations of FF androstenedione in E-I follicles 5-fold. Granulosa cells from preovulatory follicles (Experiment 2) of heifers fed HL diets secreted 2.1- to 3.5-fold greater (p less than 0.03) quantities of pregnenolone and progesterone in vitro. Finally, feeding HL supplements to postpartum range cattle for 30 days increased (p less than 0.05) the incidence of ovarian luteal activity by 18% (Experiment 3). Shifts in lipid metabolic status modify reproductive potential in cattle, independently of dietary energy intake.

Using embryonic stem cells to introduce mutations into the mouse germ line.

Abstract: It is now possible, through the use of a number of experimental technologies, to transfer genetic information into mouse embryos to stably alter the genetic constitution of mice, This experimental approach, namely the generation of so.termed “transgenie” animals, is affording new insights into a wide variety of biological problems. This review focuses on one system for the generation of transgenic mice, which utilizes tissue culture cell lines of embryonic stem cells, termed ES cells. The remarkable property of ES cells is that they retain the potential to reform an embryo; when they are replaced inside a carrier embryo, they resume normal development and contribute to all the tissues of the live-born chimeric animal. Recent experiments, using a repertoire of gene transfer techniques, have shown that ES cells are amenable to a variety of experimental manipulations in tissue culture. Moreover, it has been demonstrated that these genetically altered cells can be transferred into the germ line of chimeric mice, thus allowing the production of unique strains of animals for study. The applications of the ES cell system are reviewed, with particular emphasis on their use for the generation of random insertional mutations using a retrovirally mediated mutagenesis approach. Finally, the use of ES cells in conjunction with the recently described technique of homologous recombination, or ‘gene targeting,” is discussed. This technology allows the generation of animals carrying extremely precise genetic modifications of endogenous genes.

Expression of messenger RNAs encoding insulin-like growth factor-I, -II, and insulin-like growth factor binding protein-2 in bovine endometrium during the estrous cycle and early pregnancy.

Abstract: The present study characterized the changes in concentrations of insulin-like growth factors-I and -II (IGF-I and IGF-II) in uterine luminal flushings, and the endometrial mRNA levels of IGF-I, IGF-II, and IGF binding protein-2 (IGFBP-2) obtained from Days 0 through 18 of the bovine estrous cycle and early pregnancy. Concentrations of IGF-I and IGF-II in uterine flushings were greater on Days 0 and 5 than on other days of the estrous cycle or pregnancy. Northern blot analysis of endometrial poly(A)+ RNA revealed two major transcripts of 7.5 and 1.0 Kb for IGF-I, one major transcript of 4.0 Kb for IGF-II, and one major transcript of 1.3 Kb for IGFBP-2. RNA dot-blot analyses indicated that endometrial expression of IGF-I mRNA was unaffected by day of the estrous cycle or status (cyclic vs. pregnancy). However, endometrial expression of IGF-II mRNA was greater in pregnant than in cyclic endometrium on Days 15 and 18. Levels of endometrial IGFBP-2 mRNA increased (p less than 0.05) between Days 10 and 18 of the estrous cycle and early pregnancy. Results suggest that the presence of the bovine conceptus has a stimulatory effect on endometrial expression levels of IGF-II, whereas progesterone appears to be involved with enhancement of IGFBP-2.