Showing papers in "Biology of Reproduction in 1987"

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte.

Abstract: 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure.

Abstract: Depriving rats of luteinizing hormone (LH) causes Leydig cells to lose smooth endoplasmic reticulum and diminishes their P450 C17-hydroxylase/C17,20-lyase activity (Wing et al., 1984). LH administration to hypophysectomized rats prevents these changes in Leydig cell structure and function (Ewing and Zirkin, 1983). We adopted a multistep procedure of rat Leydig cell isolation to study the trophic effects of LH on steroidogenesis in the Leydig cell. Our method employs vascular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll gradient centrifugation. The purified Leydig cell fraction obtained after Percoll density-gradient centrifugation contains 95% well-preserved 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)-staining cells with ultrastructural characteristics of Leydig cells. These Leydig cells produced 248 and 29 ng of testosterone/10(6) Leydig cells when incubated for 3 h with and without a maximally stimulating concentration of ovine LH. Purified Leydig cells obtained from control rats and rats treated with testosterone-estradiol (T-E) implants for 4 days to inhibit LH production were incubated with a saturating concentration (2 microns) of pregnenolone. Leydig cells from control and T-E-implanted rats produced 537 and 200 ng of testosterone/10(6) Leydig cells X 3 h, respectively, suggesting a defect in the steroidogenic reactions converting pregnenolone to testosterone in Leydig cells from T-E-implanted rats. By using rabbit antibodies to the P450 C17-hydroxylase/C17,20-lyase pig microsomal enzyme, immunoblots of one-dimensional sodium dodecyl sulfate polyacrylamide gels of Leydig cell microsomal protein from control and 4- and 12-day T-E implanted rats revealed a continued loss of enzyme as the period of LH withdrawal continues. These results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.

Sperm transport and motility in the mouse oviduct: observations in situ.

Abstract: 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.

Development potential of bovine oocytes matured in vitro or in vivo.

Abstract: 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.

Steroid feedback inhibition of pulsatile secretion of gonadotropin-releasing hormone in the ewe.

Abstract: 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.

Heterogeneity of sperm nuclear chromatin structure and its relationship to bull fertility.

Abstract: 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.

Apoptosis as the mode of uterine epithelial cell death during embryo implantation in mice and rats.

Abstract: 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.

Interactions between prolactin and dopaminergic neurons.

Abstract: The secretion of prolactin from the adenohypophysis is tonically inhibited by dopamine that is released into the hypophysial portal blood from terminals of tuberoinfundibular neurons located in the external layer of the median eminence. These tuberoinfundibular neurons are unique among other dopaminergic neurons in the brain (including the well-characterized nigrostriatal neurons) in that they are not directly regulated by dopaminergic receptor-mediated mechanisms, but instead are selectively responsive to changes in prolactin concentrations in blood and cerebrospinal fluid. In the rat, the activity of the tuberoinfundibular dopaminergic neurons is higher in the female than in the male, exhibits a characteristic cyclical pattern during the first half of pregnancy and is constantly high as a result of stimulation by placental lactogen during the last 9 days of pregnancy, and is reduced in lactating animals and acutely inhibited during suckling.

Amino acid content of preimplantation rabbit embryos and fluids of the reproductive tract.

Abstract: 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.

Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos.

Abstract: 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.

Lipids of plasma membrane and outer acrosomal membrane from bovine spermatozoa.

Abstract: 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 endocrinology of puberty and reproductive functioning in female cotton-top tamarins (Saguinus oedipus) under varying social conditions.

Abstract: 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.

Initiation of hyperactivated flagellar bending in mouse sperm within the female reproductive tract.

Abstract: 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.

Endocrine mechanisms of puberty in heifers. Role of hypothalamo-pituitary estradiol receptors in the negative feedback of estradiol on luteinizing hormone secretion.

Abstract: The hypothesis tested was that the decline in negative feedback of estradiol on secretion of luteinizing hormone (LH) that occurs as puberty approaches in heifers results from a decline in the number of receptors for estradiol in the hypothalamus and/or pituitary. In addition, associated changes in receptors for luteinizing hormone-releasing hormone (LHRH) in the pituitary, ovarian follicle development, and uterine growth were characterized. Fifty prepubertal heifers, 234 to 264 days of age, were used. Six heifers of median body weight were designated controls, and sequential blood samples were collected at 20-min intervals for 24 h every 2 wk from 249 days of age through puberty and analyzed for concentrations of LH. Frequency of LH pulses/24 h was regressed on number of days prepuberty to develop a prediction equation for puberty. Thirty of the remaining 44 heifers were killed at 253, 302, and 351 days of age (n = 10/group), and tissues for described analyses were collected. Three to 5 days before tissue collection, sequential blood samples were obtained from these heifers, as described for control heifers to determine frequency of release of LH. With this information, number of days prepuberty at the time of tissue collection was estimated from the prediction equation developed with data from control heifers. The average age at puberty in control heifers was 366 days. The average age at puberty of heifers that were not killed or included in the control group (n = 14) was 360 days. Receptor and morphological data were related to the estimated onset of puberty. Cytosolic concentration of receptors for estradiol (fmoles receptor/mg cytosolic protein) in the anterior hypothalamus, medial basal hypothalamus, and anterior pituitary declined (p less than 0.05) as puberty approached. No change in concentration of receptors for estradiol was observed in the stalk median eminence or preoptic area. The concentration of receptors for LHRH in the anterior pituitary did not change as puberty approached. Uterine weight increased rapidly during the 50 days preceding puberty. The number of small, medium, or large follicles and the wet, pressed, or dry weight of the ovaries did not change as puberty approached. Follicles with a diameter greater than 12 mm were found only in the 3 heifers estimated to be closest to puberty at the time of tissue collection. The hypothesis that the decline in estradiol feedback on secretion of LH during the prepubertal period in heifers may result from a decline in the concentration of binding sites for estradiol at the hypothalamus and/or pituitary is supported by this study.

The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content.

Abstract: 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 mm to deco ndense. 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 mm of microinjection. Caput epididymal sperm nuclei, with intermediate S-S bond content, decondensed in 10-20 mm. 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 of sperm nuclear decondensation and pronucleus formation depends in part upon the S-S bond content of the sperm nucleus.

Uptake of an oviductal antigen by the hamster zona pellucida.

Abstract: 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.

Time-course and steroid specificity of aromatase induction in rat hypothalamus-preoptic area.

Abstract: 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.

Hypoxanthine causes a 2-cell block in random-bred mouse embryos.

Abstract: 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.

In vitro regulation of pig Sertoli cell growth and function: effects of fibroblast growth factor and somatomedin-C.

Abstract: 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.

Similarity in ejaculate-endocrine characteristics in captive versus free-ranging cheetahs of two subspecies.

Abstract: Ejaculate-endocrine characteristics were measured in 23 captive cheetahs (Acinonyx jubatus jubatus) in North American zoos and in 8 free-ranging cheetahs (A. j. raineyi) in eastern Africa (Tanzania). A standardized electroejaculation protocol was used, and numbers of motile spermatozoa were similar (p>O.05) between groups. Of the spermatozoa collected by electroejaculation, 70.6 ± 3.3% and 75.9 ± 4.4% were morphologically abnormal in the captive “North American” and in the free-ranging, eastern African populations, respectively. Adrenal activity, as measured by an acute, temporal rise and fall in serum cortisol levels during and after electroejaculation, was no different (p> 0.05) between groups. Although serum luteinizing hormone (LH) levels were less (p< 0.05) In the free-ranging than in the captive animals, serum testosterone concentrations were similar. The data indicate that the comparatively poor reproductive performance of cheetahs maintained in zoological parks is not attributable to a captivity-induced response afflicting the male. Furthermore, there is no evidence that ejaculate/endocrine characteristics differ between the two subspecies. Because adrenal /gonadal activity and the number of pleiomorphic spermatozoa are similar between the test groups, the results suggest that spermatozoal diversity originates as a result of the extreme genetic monomorphism observed universally in the species.

The molecular basis of gonadotropin-releasing hormone (GnRH) action in the pituitary gonadotrope.

Abstract: GnRH is a hormone whose time has come. In the less than 15 years since the structure of gonadotropin-releasing hormone (GnRH) was announced, thousands of analogs have been prepared, and we have learned a great deal about the molecular basis of action of this hormone; this information has been employed to identify ways that GnRH can be used to benefit humanity. GnRH can be used to promote or inhibit fertility in men and women, and to treat steroid-dependent neoplasia, precocious puberty, cryptorchidism, and other diseases. In addition, GnRH has proven to be useful in veterinary medicine and for synchronized breeding of fish for food. The target sites for GnRH are limited; this has resulted in the production of highly specific agents with minimal side effects. The large number of agonist and antagonist analogs have provided an impressive arsenal for clinical work and greatly increased the capacity to design experiments; they are in large part responsible for the rapid progress in this

Inhibition of testicular testosterone biosynthesis following experimental varicocele in rats.

Abstract: In an attempt to determine whether defective testicular testosterone (T) biosynthesis may be associated with a varicocele, an experimental study was performed in adult rats whereby a unilateral left varicocele was surgically created. At 2, 4, 8, and 12 wk following the creation of the varicocele, intratesticular T as well as the activities of three (17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase) of the five enzymes in the delta 4 pathway of testicular T biosynthesis were measured. Intratesticular T (ng/g testis +/- SEM) in the left testis decreased significantly from 121 +/- 21 in the control group to 59 +/- 8 in the two-wk varicocele group (p less than 0.01), and remained significantly suppressed throughout the experimental period. The T concentrations in the right testis paralleled those in the left in both the control and varicocele animals. At 2 wk following the creation of the varicocele, the activity (nmol/min/testis +/- SEM) of the 17,20-desmolase enzyme decreased significantly, from 115 +/- 8 in the left testis of control rats to 87 +/- 6 in the left testis of the varicocele animals (p less than 0.025), and remained low throughout the 12 weeks of the study. The activity of the 17 alpha-hydroxylase enzyme was significantly decreased at the 8th and 12th weeks of the study, while the 17 beta-hydroxysteroid dehydrogenase activity did not show any significant change during the study period. The enzyme activities in the right testis paralleled those in the left testis.(ABSTRACT TRUNCATED AT 250 WORDS)

Stage-dependent levels of specific mRNA transcripts in Sertoli cells.

Abstract: Localization and stage-dependent levels of transfewin and sulfated glycoprotein-2 (SGP-2) mRNAs were examined in rat testes by in situ and soluble hybridization of mRNA with a single-stranded RNA probe prepared with the SP6.5 vector. Biotinylated RNA probes were identified in testicular tissue by using a biotinylated glucose oxidase-avidin system followed by a treatment with an appropriate electron carrier and a tetrazolium salt. This procedure demonstrated that the anatomical site of transferrin and SGP-2 gene expression was the Sertoli cells. Tritium-labeled RNA probes were visualized by radioautography. Negative and positive controls as well as in situ hybridization in Sertoli and myoid cells in culture indicated again that the cytoplasm of Sertoli cells was the anatomical site of transferrin and SGP-2 expression. Quantitative radioautography revealed cyclic variations in the level of both transferrin and SGP-2 mRNAs. The level of transferrin mRNA was relatively high from Stage I to Stage VIII. At Stage IX, the level decreased acutely and remained low in Stage X. The level of transcripts increased dramatically at Stage XlIl, remaining high until Stage XIV. In the case of SGP-2 mRNAs, levels of transcripts were similar in most stages except at Stages VII and VIII, where higher levels were observed. These data were substantiated by similar results obtained by solution hybridization of both recombinant cRNAs with m RNAs from selected seminiferous tubules staged by transillumination. Thus, our results demonstrated a stage-specific regulation of transfewah and SGP-2 mRNA levels in Sertoli cells. . I NT RO DU CTJ ON

Distribution and number of spermatozoa in the oviduct of the golden hamster after natural mating and artificial insemination.

Abstract: 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.

Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: electron microscopic localization.

Abstract: 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.

Changes in content of cytochrome P450(17)alpha, cytochrome P450scc, and 3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarian follicles and corpora lutea: correlation with theca cell steroidogenesis.

Abstract: 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.

Hormonal control of sexual behavior in the female rat: molecular, cellular and neurochemical studies.

Abstract: The present era of molecular research on steroid hormone action originates from the demonstration that there are intracellular protein receptors that bind steroid hormones and carry them to sites in cell nuclei where they regulate gene expression (Jensen and Jacobson, 1962; Toft and Gorski, 1966; Jensen et al., 1968; O’Malley and Means, 1974). Researchers of steroid hormone action in brain tissue have taken advantage of this revolution in biochemistry and cell biology and have found a way to understand the actions of gonada! and adrenal steroids on neural cells in cellular and molecular terms. However, the road has not been an easy one. The difficulties stem from the extreme complexity and anatomical heterogeneity of the brain, and the precise and highly localized neural networks involved in governing behavior. There also have been conceptual problems caused by a common tendency to regard the hormone as a stimulus and the behavior it regulates as a response, a relationship that implies temporal immediacy. However, because a hallmark of steroid hormone-regulated behaviors is their long latency and duration in relation to hormone priming, it has been necessary to look at the role of hormones in a different light. More than 40 years ago, the preeminent pioneer in elucidating hormones’ effects on behavior, Frank Beach, anticipated the current view of steroid hormone action by writing:

Analysis of Qa-2 antigen expression by preimplantation mouse embryos: possible relationship to the preimplantation-embryo-development (Ped) gene product.

Abstract: The preimplantation-embryo-development (Ped) gene, a gene that controls the cleavage rate of preimplantation mouse embryos, maps to the Qa-2 subregion of the mouse major histocompatibility complex (MHC). A highly sensitive enzyme-linked immunosorbent assay (ELISA) procedure was used to detect Qa-2 antigens on mouse embryos. The use of a monoclonal antibody specific for Qa-2 antigens showed that Qa-2 antigens were present on oocytes, 2-cell, 8-cell, and blastocyst-stage embryos, with the greatest expression found on blastocysts. Expression of Qa-2 antigens by the embryos correlated completely with Ped gene phenotype. Those embryos expressing the fast Ped allele showed the presence of Qa-2 antigens (Qa-2a mice), whereas those embryos expressing the slow Ped allele showed the absence of Qa-2 antigens (Qa-2b mice). It is hypothesized that the Qa-2 antigen may be the Ped gene product.

Purification, secretion and immunocytochemical localization of the uterine milk proteins, major progesterone-induced proteins in uterine secretions of the sheep.

Abstract: Restriction of the conceptus to one uterine horn of the pregnant ewe results in the accumulation of fluid called uterine milk (UTM) in the contralateral horn. Two basic polypeptides, called the uterine milk proteins (UTM-proteins; Mr = 55,000 and 57,000 as determined by polyacrylamide-gel electrophoresis using sodium dodecyl sulfate), accounted for the majority of the protein in uterine milk. The two UTM-proteins were glycoproteins and were readily purified from uterine fluids by cation-exchange chromatography on carboxymethyl (CM)-cellulose followed by Sephacryl S-200 gel-filtration. The purified UIM-proteins had a weight-average molecular weight of 50,700± 4,200, as determined by equilibrium sedimentation analysis. Endometrial explants from pregnant ewes were cultured in the presence of radioactive amino acids and released UTM -proteins into the medium as their major secretory products. The UTM-proteins were secreted into the uterine lumen of nonpregnant, ovariectomized ewes given daily injections of progesterone. Estrone alone was ineffective in inducing UTM -protein production. Immunocytochemicalstudies indicated that synthesis of the UTM -proteins was confined to the surface and glandular epithelium of the uterus.

Role of 4-hydroxylated estradiol in reducing Ca2+ uptake of uterine arterial smooth muscle cells through potential-sensitive channels.

Abstract: Entry of ionic Ca2 + into the vascular smooth muscle cell for contraction is thought to be mediated by two major membrane channels. The first are designated as potential-sensitive channels (PSCs), which are opened by membrane depolarization, and the second, as receptor-operated channels (ROCs), which are activated by csl-receptor-ligand interactions. This study was designed to determine the presence of these 2 distinct populations of Ca2 + entry channels in smooth muscle cells of the uterine arteries in pigs. This was studied by measuring the baseline tone and contractile properties of uterine arteries in in vitro perfusion studies, as well as their specific Ca2 + uptakes. These parameters showed markedly different sensitivities towards two smooth muscle inhibitors used in this study: D-600 and amrinone. D-600 specifically inhibits uptake of extracellular Ca2 + through PSCs, while amrinone specifically inhibits Ca2 + uptake through ROCs. By choosing an appropriate concentration of D-600 or amrinone, Ca2 + uptake and contractions of uterine arterial segments induced by high-K� (PSC activator) and phenylephrine (ROC activator) could be selectively inhibited. Furthermore, it was demonstrated that the blockade of Ca2 + uptake by D-600 and a,nrinone was additive, excluding the interpretation of a common Ca2 + pathway with two separate mechanisms for opening it. It was also determined that 4-hydoxylated estradiol (40H-E2), a compound known to increase uterine blood flow in pigs, decreased Ca2 + uptake through the PSCs and exhibited no effect on ROCs. The presence of separate Ca2 + pathways that can be activated independently by agonists may indicate a refined system for controlling uterine blood flow.