Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.
Bovine oocytes matured in vivo or in vitro were evaluated after sperm-oocyte incubation for frequency of sperm penetration, frequency of male pronuclei formation, and embryonic development. The frequency of sperm penetration was not different for in vitro matured oocytes (216/295, 73%) vs. in vivo matured oocytes (119/176, 70%). However, formation of male pronuclei was reduced (p less than 0.05) for oocytes matured in vitro (149/216, 69%) vs. in vivo (104/119, 88%). Early embryonic development was evaluated 48 h after the onset of sperm-egg incubations. In vitro matured and fertilized oocytes failed to develop to the 2-cell stage (3/88, 3%), whereas oocytes matured in vivo showed normal development (23/56, 40%) to the 2- and 4-cell stage. Development to the blastocyst stage was evaluated after 5 days in ovine oviducts (in vivo). Morulae and blastocysts were obtained only after in vitro fertilization from oocytes that were in vivo-matured (recovered from oviduct, 14/56, 25%; recovered from follicle, 36/80, 45%). Oocytes that were matured in vitro and fertilized in vitro failed to develop to morulae (0/33) in vivo.
Sperm transport and motility were studied through the transparent walls of the mouse oviduct by direct microscopic observation and videomicrography. Observations were made on excised female tracts 1-2 h post-coitus (pc) and 1-2 h before and after the approximate time of ovulation. Motile sperm were seen at the uterine entrance to the uterotubal junction (UTJ) in all females at 1-2 h pc, but in fewer females at later times. The intramural UTJ was usually constricted and held few sperm. The extramural UTJ and adjacent lower isthmus contained many motile sperm at 1-2 h pc. Apparently, the column of sperm moved upwards because in some females, sperm were found in the upper isthmus and not in the UTJ at the later time points. Few sperm were seen in the ampulla in the periovulatory period, and none at 1-2 h pc. There appeared to be two mechanisms retaining sperm in the lower oviduct: immobilization and adherence to the epithelium. Columns of immotile sperm were seen in the lower isthmus of some females. Motile sperm usually appeared to adhere by their heads to the oviductal epithelium, only occasionally breaking free to move vigorously about the lumen.
The long-term negative feedback effects of sustained elevations in circulating estradiol and progesterone on the pulsatile secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) were evaluated in the ewe following ovariectomy during the mid-late anestrous and early breeding seasons. GnRH secretion was monitored in serial samples of hypophyseal portal blood. Steroids were administered from the time of ovariectomy by s.c. Silastic implants, which maintained plasma concentrations of estradiol and progesterone at levels resembling those that circulate during the mid-luteal phase of the estrous cycle; control ewes did not receive steroidal replacement. Analysis of hormonal pulse patterns in serial samples during 6-h periods on Days 8-10 after ovariectomy disclosed discrete, concurrent pulses of GnRH in hypothalamo-hypophyseal portal blood and LH in peripheral blood of untreated ovariectomized ewes. These pulses occurred every 97 min on the average. Treatment with either estradiol or progesterone greatly diminished or abolished detectable pulsatile secretion of GnRH and LH, infrequent pulses being evident in only 3 of 19 steroid-treated ewes. No major seasonal difference was observed in GnRH or LH pulse patterns in any group of ewes. Our findings in the ovariectomized ewe provide direct support for the conclusion that the negative-feedback effects of estradiol and progesterone on gonadotropin secretion in the ewe include an action on the brain and a consequent inhibition of pulsatile GnRH secretion.
The relationship between sperm nuclear chromatin structure and fertility was evaluated in two groups of Holstein bulls: Group 1, 49 mature bulls, and Group 2, 18 young bulls. Fertility ratings had been estimated for Group 1 and nonreturn rates were known for Group 2. Semen samples were measured by the sperm chromatin structure assay (SCSA): sperm were treated to induce partial in situ DNA denaturation, stained with acridine orange, and evaluated by flow cytometry. Acridine orange intercalated into double-stranded DNA emits green fluorescence upon excitation with 488 nm light, and red fluorescence when associated with single-stranded DNA. An index of DNA denaturation per cell is provided by alpha-t [alpha t = red/(red + green) fluorescence]. The standard deviation (SD alpha t), coefficient of variation (CV alpha t) and proportion of cells outside the main population (COMP alpha t) of the alpha t distribution quantify the extent of denaturation for a sample. Intraclass correlations of the alpha t values were high (greater than or equal to 0.70), based on four collections obtained over several years from Group 1 bulls. Negative correlations were obtained between fertility ratings and both SD alpha t (-0.58, p less than 0.01) and COMP alpha t (-0.40, p less than 0.01) in Group 1, and between nonreturn rates and both SD alpha t (-0.65, p less than 0.01) and COMP alpha t (-0.53, p less than 0.05) in Group 2. These data suggest that the SCSA will be of value for identification of low fertility sires and poor quality semen samples.
An ultrastructural study of mouse and rat embryo implantation sites was undertaken to determine whether the uterine luminal epithelial cells surrounding the blastocyst exhibited the morphologic characteristics of apoptotic or necrotic cell death. In both species the epithelial cells exhibited all of the characteristics of apoptosis, including surface blebbing, shrinkage and fragmentation of the cells, condensation of chromatin, and indentation and fragmentation of nuclei. Cytoplasmic organelles remained morphologically intact, and the cytoplasm maintained normal or increased staining density. Also, the epithelial cells and cell fragments were phagocytosed by the adjacent trophoblast cells. The epithelial cells did not exhibit the characteristics of necrotic cell death, such as swollen cells and mitochondria, damaged surface membranes, and disintegrated cytoplasmic organelles. We conclude that uterine epithelial cells surrounding mouse and rat embryos during implantation undergo apoptotic cell death leading to their phagocytosis by trophoblast cells.
We studied the time-course and steroid specificity for aromatase induction in the hypothalamus-preoptic area (HPOA) of the adult male rat. Aromatase activity (AA) was measured in tissue homogenates by using a radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H] androstenedione. We found that by 48 h after administration of testosterone, HPOA AA was significantly (p less than 0.01) greater than control values in castrated rats. In contrast, AA was significantly (p less than 0.01) reduced 12 h after castration, and reached its lowest levels by 4 days after castration. Several other steroids, administered in 3-cm Silastic capsules for 7 days, were tested for their capacity to induce hypothalamic AA. In addition to testosterone, only 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were effective. Neither the stereoisomers of these compounds nor several other steroids, including estradiol, progesterone, and corticosterone, were active. This profile of activity indicates that the induction of HPOA AA is androgen-specific and, together with the demonstrated time-course of induction, lends further support to the hypothesis that androgens regulate AA through a receptor mechanism and the synthesis of new protein.
Between 1 and 4% of human oocytes fertilized in vitro are tripronuclear. It has been reported that these tripronuclear oocytes can develop to grossly normal-appearing morulae and that chromosomally, these embryos could be triploid, diploid, or severely depleted. The etiology and proportion of apparently diploid and aneuploid embryos deriving from tripronuclear human oocytes is unknown. This study provides evidence for the first time that most (18 of 29) tripronuclear human oocytes cleave directly to 3-cells at the first cleavage division. These embryos have a severely abnormal (but not triploid) chromosomal complement. Furthermore, some (4 of 29) tripronuclear human oocytes cleave to 2-cells plus an extrusion, and these embryos are diploids, whereas some (7 of 29) cleave to 2-cells, and these embryos are triploid after the first cleavage division. These findings demonstrate that most tripronuclear human oocytes have an altered cleavage pattern at the first cleavage division, that most tripronuclear human oocytes (76% in this study) do not develop into triploid embryos, and that a correlation exists between the pattern of the first cleavage division and the subsequent karyotype of these embryos. Insight into the mechanisms by which these oocytes fail to develop into triploid embryos is also provided.
Ham's F-10, a chemically defined, complex culture medium, commonly used for in vitro fertilization of human as well as animal oocytes, blocked development at the 2-cell stage of greater than 92% of embryos from random-bred Swiss mice (CD-1), but did not block development of embryos from hybrid-inbred mice (BDF1). In contrast, BWW, a simple, modified Kreb's-Ringer bicarbonate medium, supported development to blastocysts of 85% and 100% of 2-cell embryos from CD1 and BDF1 females, respectively. As little as 15% (v/v) Ham's F-10 added to the BWW blocked the development of the random-bred embryos. Supplementing the BWW with Ham's F-10 components revealed that hypoxanthine (6-30 microM) was responsible for the developmental block to the random-bred embryos. The hypoxanthine block was partially (40%) reversed by adding the chelating agent, ethylenediaminetetraacetic acid. Breeding experiments showed that the hypoxanthine sensitivity of embryos from CD-1 mothers was not affected by the paternal genome.
As a companion to amino acid transport and protein synthetic studies, it was of interest to quantify the amino acid pools in embryos and reproductive tract fluids during preimplantation development. Primary amines in the acid-soluble extracts of embryo and fluid samples were separated by high-performance liquid chromatography, reacted with o-phthalaldehyde, and quantified by fluorescence emission. The amino acid compositions of embryos were like those of corresponding reproductive tract fluids. Taurine was high in eggs and fluids but declined with development, while glycine levels rose. Glycine was highest in concentration in all samples (except the egg), followed by glutamate and alanine, while most other amino acids were consistently of low abundance.
Sexual maturation and fertility were assessed in fourteen cotton-top tamarin (Saguinus oedipus) females under various social conditions. Six tamarin females (20-28 mo of age) showed a suppression of fertility while living with their families. Hormonal profiles demonstrated low, acyclic levels of urinary luteinizing hormone (LH) and estrone-conjugates (E1C). A rapid onset of ovarian and pituitary cyclicity occurred when four of the six females were removed from their families and paired with an unrelated male. In one female, an ovulatory LH peak occurred as early as eight days after pairing and resulted in conception and full-term pregnancy. Two of the six females were housed in total isolation for 30 days following their removal from the family and prior to pairing. Gradual increases in hormone concentrations occurred during isolation; however, there was no ovarian cyclicity until each female was paired with an unrelated male. In all six females, conception occurred before or as a result of the third ovulatory cycle. Partial isolation of a 36-mo-old female resulted in elevated LH and E1C levels, but cyclicity was not observed until the female was paired with an unrelated male. These findings indicate that removal of a female from the family alone does not initiate ovarian cycling. Sexual maturation, or puberty, occurs in female tamarins living with their families between 15 and 17 mo of age when mean LH and E1C levels began to increase. However, when a female is removed and paired at 9 mo of age with an unrelated male, elevated levels of LH and E1C may be seen by 10 and 11 mo of age. Our findings indicate that a suppression of fertility occurs in cotton-top tamarins living with their families, but that reproductive suppression does not affect the process of sexual maturation. Both removal from the family environment and stimulation by an unrelated male tamarin were necessary to induce normal reproductive activity. An acceleration of puberty occurred when a female tamarin was removed from her family early in development and paired with a male.
A group of female hamsters was mated with males of proven fertility either several hours before or during ovulation. Another group of females was artificially inseminated several hours before ovulation. Females were killed at various times after the onset of mating or artificial insemination, oviducts were fixed and sectioned serially, and spermatozoa were counted individually as to their location in the oviduct. Regardless of the type or time of insemination, the vast majority of spermatozoa that entered the oviduct remained in the lower segments of the isthmus (the intramural and caudal isthmus) without ascending to the ampulla. The lower segments of the oviduct, particularly the caudal isthmus, appeared to be acting as a "sieve" and/or "sperm reservoir." In females mated or artificially inseminated prior to ovulation, virtually no spermatozoa reached the cephalic isthmus or ampulla until the commencement of ovulation. Although a few spermatozoa reached the ampulla by 1 h after the onset of mating, they were the exception rather than the rule. When females were mated during ovulation, spermatozoa spent a minimum of about 3 h in the caudal isthmus before ascending to the ampulla. The number of spermatozoa that entered the oviduct after artificial insemination was considerably lower than in naturally mated animals, but this low number was apparently large enough to ensure complete fertilization.
Plasma membrane (PM), primarily from the anterior sperm head, and outer acrosomal membrane (OAM), were isolated from ejaculated bovine spermatozoa, and the major lipid classes were characterized. Whole sperm (WS) lipids were analyzed for comparison. PM was removed by nitrogen cavitation and purified by sucrose density-gradient centrifugation. The OAM was removed by centrifugation through hyperosmotic sucrose and recovered by sucrose density-gradient centrifugation. The PM contained primarily spherical vesicles from the region overlying the OAM and was enriched 9- and 13-fold in 5'-nucleotidase and alkaline phosphatase activity, respectively, compared to the original cavitate. The OAM was recovered as caplike structures with associated ground substance. Protein, phospholipid, and cholesterol (PR, PL, and CH as micrograms/5 x 10(9) sperm) were 300, 467, and 93 for PM and 276, 111, and 25 for OAM, respectively. Corresponding values for WS (mg/5 x 10(9) sperm) were 31.4, 6.63, and 0.72. The PR/PL (w/w) and CH/PL (mol/mol) ratios were 0.66 and 0.38 for PM; 2.48 and 0.26 for OAM; and 4.39 and 0.22 for WS. Cholesterol was the only free sterol detected by gas/liquid chromatography in WS, PM, and OAM, with traces of CH sulfate present in all three preparations. Glycolipid tentatively identified as sulfogalactolipid was detected by thin-layer chromatography (TLC) in PM but not OAM. Phospholipid composition of WS and membranes was determined by TLC. Cardiolipin (3% of total PL) was present in WS only. Choline, ethanolamine, and inositol phosphoglycerides (CP, EP, PI, PIP, PIPP); sphingomyelin (SP); phosphatidylserine (PS); and lysophosphatidylcholine (LPC) were present in WS, PM, and OAM. Approximately 50% of total PL was CP in all preparations; SP was 13% of PL in PM and 17% in OAM (p less than 0.05); EP was 7% of PL in PM and 10% in OAM (p less than 0.05). The differences in composition between PM and OAM is discussed with respect to capacitation and ability of sperm to undergo the acrosome reaction.
The effects of insulin, somatomedin-C (Sm-C), epidermal growth factor (EGF), fibroblast growth factor (FGF), vitamin E, and retinoic acid on growth and function of immature cultured pig Sertoli cells were investigated. All these factors, except vitamin E, stimulated Sertoli cell DNA synthesis and proliferation. The mitogenic effects of insulin observed only at micromolar concentrations were similar to those induced by nanomolar concentrations of Sm-C or EGF, but significantly less than those induced by FGF. The effects of EGF and Sm-C were almost additive, whereas those of Sm-C and FGF were synergistic. After a 6-day treatment, FGF and retinoic acid induced a significant increase in the number of follicle-stimulating hormone (FSH) receptors per cell, and in FSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production. Sm-C, which alone had no effect on these two parameters, potentiated FGF action. Basal plasminogen activator activity was enhanced after the 6-day treatment with EGF plus insulin and, particularly, with FGF plus insulin. Similarly, the response of the latter group to FSH was significantly higher than in any other group of cells. FGF was also able to stimulate cell multiplication and enhanced the FSH receptor number of Sertoli cells isolated from 15- and 26-day-old rats. Thus, FGF is the most potent known mitogenic factor for cultured Sertoli cells, and it stimulates the phenotypic expression of these cells.
Using an antiserum raised against hamster oviductal zona pellucida, we observed specific immunogenic components of the reproductive tract on the zonae of oviductal eggs and in oviductal fluid. Results of immunohistochemical studies suggested that these oviductal components may originate from epithelial cells of the isthmus and, to a lesser extent, of the ampulla and fimbria. The oviductal immunogenic components have also been observed within the bursal cavity, which contains the ovary. These observations suggest that these oviductal components may play an important role in the first steps of the hamster reproductive process.
The following study was undertaken to determine which hormones (luteinizing hormone, LH, and prolactin, PRL) and enzymes (cytochrome P450(17)alpha, nicotinamide adenine dinucleotide phosphate [NADPH]-cytochrome P450 reductase, 3-hydroxy-3-methylglutaryl [HMG] CoA reductase, cholesterol side-chain cleavage cytochrome P450 [P450scc], and adrenodoxin) were associated with the regulation of androgen biosynthesis by developing rat follicles and corpora lutea in vivo as well as by thecal explants maintained in culture. Immunoblots of soluble cell extracts of small antral (SA), preovulatory (PO), and luteinizing (PO + human chorionic gonadotropin [hCG], 7 h) follicles, newly formed corpora lutea (PO + hCG, 24 h), and corpora luteal isolated on Day 15 of pregnancy, demonstrated that cytochrome P450(17)alpha was low in SA follicles, selectively increased 4-fold in PO follicles, and decreased to less than 10% within 7 h after hCG. Filter hybridization assays using a 32P-labeled cytochrome P450(17)alpha cDNA probe demonstrated that changes in the content of P450(17)alpha mRNA exhibited a pattern similar to that of the enzyme. Conversely, immunoblots for other microsomal enzymes either exhibited no change (NADPH cytochrome P450 reductase) or a transient increase after the hCG surge (HMG CoA reductase), whereas the mitochondrial enzymes either increased markedly in association with luteinization (cytochrome P450scc) or were increased in a more transient manner (adrenodoxin). The LH-induced loss of cytochrome P450(17)alpha in vivo was not associated with loss of androgen biosynthesis when luteinizing theca were placed in culture in medium containing either LH or LH and PRL, suggesting that other hormones, or the presence of other cell types, are required to maintain the decrease in cytochrome P450(17)alpha in vivo. Conversely, the LH-induced increase in cytochrome P450scc in vivo was associated with the maintenance of elevated progesterone production by theca in culture, suggesting that cytochrome P450scc may be constitutively expressed in luteinized theca. Thus, thecal cell cytochrome P450(17)alpha and the regulation of its content and mRNA by LH are pivotal to the biosynthesis of androgens, the obligatory precursors for estradiol biosynthesis and the consequent development of preovulatory follicles. The molecular basis for the different effects of low versus elevated concentrations of LH on cytochrome P450(17)alpha, as well as cytochrome P450scc, remain to be determined.
To determine where and when hyperactivation is initiated in vivo, the flagellar curvature ratios (fcr) of mouse sperm within the female reproductive tract were measured from videotape recordings and compared with those of epididymal sperm incubated under capacitating conditions in vitro. The fcrs and linearities of trajectory were significantly lowered after 90 min of incubation in vitro, indicating that hyperactivation had been initiated by that time. The flagellar curvature ratios of sperm at the colliculus tubarius, within the uterotubal junction, and in the isthmus, measured at 1-2 h postcoitus and approximately 1 h before and 1 h after ovulation, were found to have fcrs that were not different from those of sperm incubated for 90 min in vitro. It was concluded that the tract sperm had initiated hyperactivated flagellar bending before the time of ovulation and before entering the oviduct. Only sperm in the lower isthmus 1 h before ovulation had fcrs that were significantly different from sperm incubated for 90 min in vitro, but not from sperm measured at the beginning of incubation in vitro. This could be the result of motility suppression in the lower isthmus.
The relationship between the timing of both sperm nuclear decondensation and male pronucleus formation in the oocyte and the relative level of disulfide bonds within the sperm nucleus was evaluated. Since reduction of sperm nuclear disulfide (S-S) bonds is a prerequisite for sperm nuclear decondensation in vitro and in vivo, we hypothesized that sperm nuclei with relatively few S-S bonds would require less time to decondense in the oocyte than sperm nuclei with higher numbers of S-S bonds, and that male pronucleus formation would occur more rapidly as well. Four types of hamster sperm nuclei, in which the extent of S-S bonding differed, were microinjected into hamster oocytes, and the time course of sperm nuclear decondensation and male pronucleus formation was charted. Cauda epididymal sperm nuclei, which are rich in S-S bonds, required 45-60 min to decondense. In contrast, nuclei containing few S-S bonds (namely sonication-resistant spermatid nuclei and cauda epididymal sperm nuclei treated in vitro with the S-S bond-reducing agent dithiothreitol) decondensed within 5-10 min of microinjection. Caput epididymal sperm nuclei, with intermediate S-S bond content, decondensed in 10-20 min. Regardless of when decondensation occurred, formation of the male pronucleus never preceded that of the female pronucleus, which occurred 1.25-1.5 h after microinjection. However, sperm nuclei with few S-S bonds were more likely than S-S rich nuclei to transform into male pronuclei in synchrony with the formation of the female pronucleus. We conclude that the timing sperm nuclear decondensation and pronucleus formation depends in part upon the S-S bond content of the sperm nucleus.
The distribution of laminin, type IV collagen, heparan sulfate proteoglycan, and fibronectin was investigated in the rat testicular lamina propria by electron microscopic immunocytochemistry. Distinct patterns were observed for each antigen within the extracellular matrix (ECM) layers of the lamina propria. Laminin, type IV collagen, and heparan sulfate proteoglycan all localized to the seminiferous tubule basement membrane. Type IV collagen and heparan sulfate proteoglycan, but not laminin, localized to the seminiferous tubule side of the peritubular myoid cells. All four of the antigens were localized between the peritubular and lymphatic endothelial cells. Failure to localize fibronectin in the ECM layer between the Sertoli and peritubular myoid cells tends to support the concept that adult Sertoli cells do not produce this protein in vivo. Intracellular immunostaining was insufficient to allow unambiguous identification of the cellular source of any of the ECM molecules.