Biomedical subjects

J K Hodges

Publications and source records attributed to J K Hodges.

At least 37 records · Page 2Linked to original sources

PubMed1997

Birth of offspring following artificial insemination in the common marmoset, Callithrix jacchus.

The objective of the study was to develop a method for artificial insemination (AI) in the common marmoset, a New World primate species. For AI to be successful, sperm must be deposited at an appropriate site and time in the female reproductive tract, details of which are currently not available for Callitrichid species. Epididymal sperm were deposited in the cervix of 18 marmoset monkeys (Callithrix jacchus) around the time of expected ovulation using either 3, 2, or 1 inseminations. Six out of 18 females conceived, resulting in the first reported births following AI in this species. These pregnancies show that the presence of coagulum in the vagina and the stimulus of the female reproductive tract by natural mating are not essential for effective sperm transport in this species. Although 3 different timing regimes for sperm deposition relative to ovulation were employed, no protocol was demonstrably better than the others in terms of number of conceptions. The proportions of motile, live, and morphologically normal sperm in the suspensions used for AI were comparable with published values for ejaculates from fertile male macaques. These preliminary results indicate that births are possible following AI in marmosets: the technique could be used to aid effective genetic management of the species and possibly to facilitate captive breeding of endangered Callitrichids.

Animals↗

PubMed1997

Development of an enzyme-immunoassay (EIA) for the measurement of follicle-stimulating-hormone (FSH) in Callitrichid primates using a monoclonal antibody against the human-FSH-beta-subunit.

Despite the importance of Callitrichid primates in both biomedical and conservation research, practical and reliable immunoassays for the measurement of follicle-stimulating hormone (FSH) have not yet been described. A panel of monoclonal antibodies against specific peptide fragments within either the alpha or beta subunit of human FSH was evaluated for their ability to recognize FSH from Callitrichid and other New World primates. One of these, monoclonal antibody 46.3h6.b7 raised against human FSH, was selected due to its ability to recognize marmoset monkey FSH and its low crossreactivity with other gonadotrophins. The antibody formed the basis of an enzymeimmunoassay using a highly purified human urinary FSH (Metrodin, Serono) preparation coupled to biotin as label and unmodified as standard. After 24 h incubation, antibody bound label was visualized by addition of streptavidin-peroxidase followed by the appropriate substrate. Parallelism was obtained between the standard and dilutions of pituitary extracts, urine and plasma from the common marmoset (Callithrix jacchus) as well as from two tamarin species (Saguinus fuscicollis and S. oedipus) and one squirrel monkey (Saimiri sciureus). Profiles of plasma and urinary FSH during the follicular phase are shown for two individual marmosets. The ability to measure FSH in Callitrichidae provides new opportunities for studies of the reproductive biology of these New World primate species.

Animals↗

PubMed1997

Binding activity of 5alpha-reduced gestagens to the progestin receptor from African elephant (Loxodonta africana).

Recent findings in the African elephant (Loxodonta africana) indicate that the major progestins contained within and biosynthesized by corpora lutea are 5alpha-reduced metabolites and that progesterone is quantitatively of minor importance. The specific gestagenic action within the reproductive tract of elephants was determined by measurement of relative binding affinity of the respective progestins to the gestagen receptor extracted from elephant endometrium. The cytosol was incubated with 40 nmol/liter [3H]ORG-2058 and increasing concentrations of the tested progestin. Progesterone (P4), 5alpha-pregnane-3,20-dione (DHP), and 5alpha-pregnane-3alpha-ol-20-one (5alpha-P-3OH) were used. The competition for binding sites on the progestin receptor was shown by decreasing counts measured after extraction with scintillation fluid. The progestin concentration which induced a 50% reduction of measured counts was estimated (C50) and relative binding affinity of progesterone to other progestins was calculated (RBA = C50progestin/C50p4). The relative binding affinity of DHP to P4 at the gestagen receptor of elephant endometrium was equivalent. The other 5alpha-reduced progestin (5alpha-P-3OH) showed no competition to the [3H]ORG-2058 receptor binding. We conclude that the biological significance of P4 and DHP at the receptor level is very similar. The higher quantitative levels of DHP in corpus luteum and serum support the hypothesis that this progestin is the major gestagen in the elephants, whereas 5alpha-P-3OH is an inactive metabolite.

5-alpha-Dihydroprogesterone↗

PubMed1997

Maturation, fertilization, and development of marmoset monkey oocytes in vitro.

This study was conducted to investigate 1) the capacity of in vitro-matured (IVM) marmoset oocytes to be fertilized and to support embryonic development in vitro and 2) oocyte meiotic maturation in relation to in vivo FSH administration, follicle size, and oocyte-cumulus cell status. Pairs of ovaries were collected on Day 4 of the follicular phase from adult females receiving either 1) human FSH (3 IU; n = 5) or 2) control (saline; n = 5) daily for 4 days. Antral follicles were excised from ovaries and separated into classes according to size: class 1 (660-840 microm), class 2 (> 840-1000 microm), class 3 (> 1000-1400 microm), and class 4 (> 1400 microm). A total of 823 partially naked and cumulus-enclosed oocytes (CEOs) were released from follicles and cultured in vitro. Cumulus cells remaining after 22 h were removed, metaphase II (MII) oocytes were inseminated with epididymal sperm, and resulting embryos were cultured until developmental arrest. Fluorescence microscopy was used to assess oocyte meiotic and embryo developmental progression. Oocyte germinal vesicle breakdown (GVB)- and MII-competencies increased significantly with follicular size (p < 0.01 and p < 0.0001, respectively), although they were independent of oocyte-cumulus cell associations. After 24 and 32 h in vitro, 69% and 93%, respectively, of CEOs with MII competence had completed meiotic maturation, and the rate of nuclear maturation increased progressively with follicle size (p < 0.01) and with the association of cumulus cells (p < 0.01). In vivo FSH priming slightly improved oocyte GVB- and MII-competencies (p < 0.01 and p < 0.05, respectively) and decreased the time required to achieve MII (p < 0.01). IVM oocytes from all follicle sizes fertilized (78-92%) in vitro, with 27% developing to morula- and 4% to blastocyst-stage embryos. This study demonstrates for the first time that IVM New World primate oocytes are able to support advanced preimplantation embryonic development in vitro. Oocyte meiotic competence and the time course of nuclear maturation are profoundly influenced by their follicular origin, and marginally by FSH treatment.

Animals↗

PubMed1997

Concentrations of progesterone and the 5 alpha-reduced progestins, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one, in luteal tissue and circulating blood and their relationship to luteal function in the African elephant, Loxodonta africana.

The 5 alpha-reduced metabolites 5 alpha-pregnane-3,20-dione (5 alpha-DHP) and 3 alpha-hydroxy-5 alpha-pregnan-20-one (5 alpha-P-3 alpha-OH) are the principal progestins biosynthesized by the African elephant corpus luteum. The aim of the present study was to determine luteal and circulating concentrations of these 5 alpha-reduced progestins in relation to progesterone (P4) and to examine whether their measurement reflects corpus luteum function. Ovarian (luteal) tissue (30 corpora lutea and 3 corpora rubra from 8 animals) and plasma samples (30 animals) were collected from pregnant and nonpregnant adult elephants shot in the Kruger National Park. Specific immunological measurement for both 5 alpha-reduced progestins and P4 was achieved by enzymeimmunoassay of tissue and plasma extracts following purification by HPLC. Mean (+/- SEM) luteal concentrations of 5 alpha-DHP and 5 alpha-P-3 alpha-OH were 79.5 +/- 9.4 micrograms/g and 196.5 +/- 24.8 micrograms/g, respectively, approximately 2-3 orders of magnitude higher than those of P4 (mean +/- SEM, 0.16 +/- 0.01 microgram/g). Whereas 5 alpha-reduced progestin concentrations tended to be lower in corpora lutea from late pregnancy compared with earlier stages and were lowest in corpora rubra, P4 levels were similar in all tissues/stages examined. The 5 alpha-reduced progestins also predominated over P4 in plasma (mean 5 alpha-DHP:P4 and 5 alpha-P-3 alpha-OH:P4 ratios 20.3 and 13.4, respectively). Similar to results for luteal tissue, plasma concentrations of 5 alpha-reduced progestins, but not of P4, were lower in late pregnancy than in earlier gestation stages and in nonpregnant animals. Moreover, plasma levels of both 5 alpha-reduced metabolites were negatively correlated with gestation age, whereas those of P4 were not. Levels of 5 alpha-reduced metabolites (without chromatography) were also measured in weekly blood samples throughout two complete ovarian cycles in one captive female. Both measurements showed a cyclic profile (similar to that of P4) with a luteal-phase elevation of 10- to 15-fold. The results indicate that 5 alpha-reduced compounds are the predominant progestins contained within and secreted by the corpus luteum of the African elephant, both during the ovarian cycle and throughout pregnancy. They also provide preliminary evidence to suggest that measurements of 5 alpha-reduced metabolites may reflect corpus luteum function more closely than those of P4.

5-alpha-Dihydroprogesterone↗

PubMed1997

A local oxytocin system is part of the luteinization process in the preovulatory follicle of the marmoset monkey (Callithrix jacchus).

Luteinization is a complex differentiation process involving the interaction of extrinsic and intraovarian factors. The aim of this study was to examine the components of an intraovarian oxytocin (OT) system during the periovulatory period in the marmoset monkey, as well as the possible relationship of these components to other factors involved in the luteinization process, using immunohistochemistry and cell culture techniques. Ovaries were collected on Day 7 of the follicular phase (before the endogenous LH surge) and on Day 8 (22 h after an exogenous hCG application, but before ovulation). Before the endogenous LH increase, OT immunoreactivity was detectable at low levels in most antral follicles, where its presence was confined to antral granulosa cell (GC) layers. In contrast, immunoreactivity for the OT receptor (OTR) was localized primarily in the basal GC layer. After application of exogenous hCG, there was a marked enhancement in both the staining intensity and the number of cells positive for OT and the OTR in all GC layers of antral follicles, especially in the preovulatory follicle. Progesterone receptor and 3beta-hydroxysteroid dehydrogenase activity in GC were clearly present only when follicles were obtained after gonadotropin stimulation. Secretion of authentic OT was demonstrated from cultured GC obtained before the LH surge, with highest amounts in cells cultured from preovulatory as opposed to smaller antral follicles. OT production could be stimulated by the application of hCG to the GC cultured from preovulatory follicles, whereas the gonadotropin was without effect on GC from small follicles. FSH had no effect on OT production by GC from either follicle type. Application of OT to the cultures caused an increase in progesterone production by GC from large preovulatory follicles but was without effect on steroidogenesis by cells from small antral follicles. These results describing the presence and distribution of OT and OTR and their modulation by hCG, as well as the luteotrophic effect of OT in cultured GC from preovulatory follicles, implicate OT as a paracrine mediator in the luteinization process in the primate ovary.

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

Reproductive efficiency in the common marmoset (Callithrix jacchus): a longitudinal study from ovulation to birth monitored by ultrasonography.

This study examines reproductive efficiency in the common marmoset monkey, Callithrix jacchus, using sequential ultrasound examinations to establish ovulation number, implantation rate, and incidence of prenatal loss. Ultrasound was carried out with a 10 or 7.5 mHz probe in nonsedated animals, daily during the late follicular phase, approximately twice a week until day 20 after ovulation, and at days 35, 56, and 85 of pregnancy to enable visualization of gestation sacs, heartbeats, and fetal heads, respectively. Ovulatory follicles could be seen 3-4 days before ovulation and by day -2, 98% of ovulating follicles were > 2mm diameter, although almost 10% of follicles of this size disappeared without ovulating. Total number of ovulating follicles for 15 females was 45 (mean ovulation rate = 3.0, range 2-4). In the 14 animals that conceived, 41 corpora lutea were identified (mean ovulation rate = 2.9) within 10 days of ovulation. All pregnancies went to term (no abortion occurred) resulting in the birth of 37 neonates (9 triplets, 5 twins) and an average litter size of 2.64. All four losses were confined to the embryonic period (< day 85), two occurring before day 35, one between days 35 and 56, and one between days 56 and 85. In demonstrating that 90% of ovulatory follicles gave rise to live offspring, the results of this study indicate an extremely high reproductive efficiency in the marmoset monkey (when maintained under favorable captive conditions) and a rate of prenatal loss much lower than that reported for other primate species.

Animals↗

PubMed1996

Relative bioactivity of endogenous luteinizing hormone during superovulation therapy for in vitro fertilization in normal-cycling women.

PURPOSE: Impaired outcome of in vitro fertilization treatment has been related to abnormal luteining hormone (LH) secretion and to the occurrence of a premature LH surge. The purpose of this study was to compare LH serum concentrations, measured by bioassay (LH-b) and immunoassay (LH-i), during early and late follicular phases in normal-ovulatory women who were stimulated with clomiphene, human menopausal gonadotropin (HMG), and human chorionic gonadotropin (hCG), while undergoing in vitro fertilization (IVF) treatment for infertility, due mainly to tubal damage. RESULTS: Of 22 patients, 15 had an LH surge (surge group) before receiving hCG and 7 did not (nonsurge group). LH-b and LH-i concentrations were higher in the late follicular phase than before clomiphene treatment, but the LH-b/LH-i (B/I) ratios remained unchanged and there were no significant differences between the LH surge and the nonsurge groups. In the surge group, the B/I ratio appeared to fall during the LH surge [presurge values, 5.4 +/- 0.5 (SD) and 5.2 +/- 0.5; surge values, 4.9 +/- 0.6 and later 4.0 +/- 0.6], but the differences were not significant, and in some individuals, the B/I ratio later rose as the LH surge progressed. CONCLUSIONS: Our findings suggest that interference with IVF outcome by a spontaneous LH surge is due to quantitative and/or exposure time effects on the oocyte, rather than to any qualitative variation in bioactivity of LH.

Adult↗

PubMed1996

Application of urinary and fecal steroid measurements for monitoring ovarian function and pregnancy in the bonobo (Pan paniscus) and evaluation of perineal swelling patterns in relation to endocrine events.

Estrone conjugates (E1C), pregnanediol glucuronide (PdG), and estriol (E3) in urine, and immunoreactive E1C, E3, pregnanediol (Pd), and progesterone (P4) in feces were determined along with records of perineal sex skin swelling throughout 7 nonconception cycles and 3 full-term pregnancies of 4 adult female bonobos (Pan paniscus). A typical preovulatory urinary E1C surge and postovulatory increase in urinary PdG were seen during the menstrual cycles. Fecal progestin levels were significantly correlated with those of PdG in urine in all cycles, while E1C measurements in feces were significantly correlated with those in urine in only 3 cycles. On the basis of hormone profiles, a variable follicular phase of 17-40 days and a relatively constant luteal phase of 11-15 days was found, resulting in cycle lengths of 31-51 days. All urinary and fecal hormones were markedly elevated during pregnancy. Measurement of E1C in both urine and feces was most useful for early pregnancy diagnosis, while E3 was of value in confirming pregnancy and assessing fetal viability. The period of perineal swelling during the cycle comprised on average 66.3% of cycle length, half of which was associated with a phase of maximum tumescence. Ovulation usually occurred within the maximum swelling phase, but timing of ovulation within this period was highly variable and was more closely associated with the end rather than the onset of maximum tumescence. The data presented here are of great practical value in the captive breeding management of bonobos and offer new opportunities for investigating basic questions of bonobo reproductive biology both in captivity and in the wild.

Animals↗

PubMed1996

Maturation of sperm motility in the epididymis of the common marmoset (Callithrix jacchus) and the cynomolgus monkey (Macaca fascicularis).

The acquisition by spermatozoa of the capacity for movement upon liberation from the epididymal duct into buffer is one of the obvious changes associated with sperm maturation and, hence, studies of sperm kinematics might provide good indices for sperm maturation. Profiles of development in the movement pattern of spermatozoa taken from six regions of the epididymis were established in marmoset and cynomolgus monkeys using computer-assisted sperm analysis. Motion of spermatozoa from initial regions was sluggish and irregular, then developed in the more distal region into erratic movement effected by an increase in the vigour of flagellation as reflected by curvilinear velocity. The tendency of immature spermatozoa to display circular motion was more common in the marmoset than in the cynomolgus monkey. In the marmoset the most drastic changes in percentage motility and kinematic parameters of spermatozoa occurred between the distal caput and the proximal corpus; maximal values for these parameters were found around the distal corpus epididymis. For the cynomolgus spermatozoa the sharpest gain in percentage motility was also achieved between the caput and proximal corpus epididymis, but with full kinematic development in the proximal cauda epididymis. These maturational changes were not synchronous among spermatozoa from the same site as they moved along the epididymis, as reflected in the heterogeneity of each sample in the kinematic properties of individual spermatozoa, especially those from the proximal corpus epididymis. The maturational profiles in the monkeys are comparable to the situation in the human, for which they may provide a useful model.

Animals↗

PubMed1995

Assessment of tissue integrity, ultrastructure and steroidogenic activity of corpora lutea of the marmoset monkey, Callithrix jacchus, following in vitro microdialysis.

Microdialysis of marmoset (Callithrix jacchus) corpora lutea in vitro was evaluated regarding morphology, activity of 3 beta-hydroxysteroid-dehydrogenase (3 beta-HSD) and progesterone (P) secretion. Two different dialysis media were used: an unbuffered ringer solution and Krebs-Henseleit buffer gassed with carbogenium. Additionally, the effects of the luteotrophin prostaglandin E2 (PGE2) on P secretion were examined for both media. In general 3 zones of tissue according to the maintenance of cellular integrity could be identified after dialysis. Structurally intact cells were found in close vicinity to the dialysis tubing or the bathing medium after 8 h of perfusion. These 2 zones were separated by a sheet of cells which showed signs of ischemic injury and whose activity of 3-beta-HSD was reduced. During dialysis with ringer solution P release stayed constantly high for a longer period of time than with Krebs buffer. With both media PGE2 stimulated P release but could not prevent the decrease in P production during dialysis with Krebs buffer. In general profiles of baseline secretion, were more stable after treatment than for untreated corpora lutea. There, under dialysis with ringer solution, the ultrastructure of cells close to the dialysis tubing was well preserved exhibiting euchromatic nuclei, tubular sER and numerous mitochondria gathered in the perinuclear region. In contrast, with Krebs buffer heterochromatization of nuclei and vesiculation of smooth endoplasmic reticulum prevailed. After application of PGE2 no histological and ultrastructural differences could be found between tissue dialysed with ringer or Krebs buffer. In these specimens the sER of zone A cells generally appeared vesiculated. Our results indicate (1) a close structure-function relationship of luteal cells in the tested system, (2) the suitability of the system to study intra-luteal regulation and (3) the necessity to control structural integrity of the dialysed tissue.

3-Hydroxysteroid Dehydrogenases↗

PubMed1995

Effect of cryoprotectants and their concentration on post-thaw survival and development of rapid frozen-thawed pronuclear stage mouse embryos.

Experiments have been conducted to develop a simple rapid-freezing protocol for pronuclear stage mouse embryos. The effect of type of cryoprotectant (dimethyl-sulphoxide (DMSO) or 1,2-propanediol (PROH)) and concentration (ranging from 3.5 to 8.0 mol/l with 0.5 mol/l sucrose) on the post-thaw morphological survival rate, on cleavage rate on development to the blastocyst stage were studied. Further, in-vivo viability of embryos frozen using the most effective cryoprotectant concentration (PROH at 7.0 mol/l) was compared with viability of non-frozen embryos. The type of cryoprotectant and its concentration influenced the survival and development of embryos to the blastocyst stage in vitro. The best development with PROH was achieved at 7.0 mol/l (66%, 128/193), whereas with DMSO the best development was achieved at 6.0 mol/l (42%, 71/171). The rates of survival and cleavage did not differ between the two best cryoprotectant concentrations (P > 0.01) but the proportion of embryos which developed to blastocyst was higher (P < 0.001) with PROH at 7.0 mol/l compared with DMSO at 6.0 mol/l. The rates of survival and development were higher (P < 0.001) with DMSO at 3.5 and 6.0 mol/l compared with similar concentrations of PROH. The cleavage and development, however, was higher (P < 0.001) at 7.0 mol/l PROH compared with the same concentration of DMSO. At 8.0 mol/l the survival and development was not different (P > 0.01) between DMSO and PROH. The rate of implantation and the percentage of live fetuses at autopsy of the recipients receiving non-frozen embryos was higher (63 and 41% respectively) than in those receiving frozen-thawed embryos (53 and 37% respectively), but not significantly different. It may be concluded that the concentration range of cryoprotectants which allows acceptable embryo viability after freezing and thawing is very narrow. The rapid protocol using dehydration in 0.25 mol/l and 0.5 mol/l sucrose followed by exposure to 7.0 mol/l PROH and 0.5 mol/l sucrose for 45 s was the most effective for cryopreservation of pronuclear stage mouse embryos.

Animals↗

PubMed1995

Meiotic competence of marmoset monkey oocytes is related to follicle size and oocyte-somatic cell associations.

This study was conducted to investigate the relationships between oocyte meiotic competence, follicle size, and occyte-somatic cell associations in the marmoset monkey (Callithrix jacchus). Follicles were excised from ovaries of nonstimulated adult cyclic females (n = 6) collected on Day 7 of the follicular phase. Follicles were separated into size groups: large preantral (260-400 microns), periantral (420-640 microns), small antral (660-1000 microns), large antral (1020-2000 microns), and preovulatory (> 2000 microns). Partially naked and cumulus/granulosa-enclosed oocytes (n = 473) were released from follicles and cultured in Waymouth's medium with 10% fetal calf serum, 1 microgram/ml human (h) FSH, and 10 micrograms/ml hLH. Somatic cells remaining after 46 h were removed, and oocytes were fixed after 48 h and mounted for viewing. Chromatin staining and microtubulin fluorescence labeling were used to assess progression of meiotic maturation and spindle normality. The follicle size distribution and oocytesomatic cell associations are reported. Competencies of oocytes to achieve germinal vesicle breakdown (GVBD) and metaphase II (MII) increased significantly (p < 0.001) with follicular size but not with the association of somatic cells. Marmoset oocytes from antral follicles resumed (GVBD) and completed (MII) meiotic maturation with high frequencies (98% and 72%, respectively), with no significant differences among size groups of antral follicles. GVBD competence was virtually absent in oocytes from preantral follicles (2%) and was acquired coincidentally with antrum formation (60%), although MII competence was attained after the completion of antrum formation. Partially naked oocytes from small antral follicles matured with a high incidence of spindle and meiotic abnormalities (44%). Marmoset oocyte meiotic competencies are notably higher than in any other nonhuman primate species studied, and a possible explanation for this phenomenon in relation to the stage of antrum formation is offered.

Animals↗

PubMed1995

Oxytocin: a follicular luteinisation factor in the marmoset monkey.

The distributions of oxytocin (OT) and the oxytocin receptor (OTR) have been characterised in preovulatory follicles of the marmoset monkey and are described in relation to the process of luteinisation using a combination of in vitro and in vivo techniques, including immunohistochemistry and granulosa cell culture. Ovaries were collected in the periovulatory phase before and 22h after exogenous gonadotropin (hCG) treatment, but prior to ovulation. Before hCG treatment, OT immunoreactivity (OT-ir) was found mostly in granulosa cells of antral follicles, especially in the layers nearest the antrum. In contrast, oxytocin receptor immunoreactivity (OTR-ir) was observed principally in basal granulosa cells of antral follicles. Progesterone receptor (PR) and 3 beta hydroxysteroid dehydrogenase (3 beta HSD), markers for luteinisation, were negative for all ovarian tissues collected before hCG treatment. After hCG treatment, almost all granulosa cell layers in antral follicles stained positively for both OT-ir and OTR-ir, most prominently in preovulatory follicles and especially in the cumulus oophorus. This increase in staining was associated with an induction of PR and 3 beta HSD activity in the granulosa cells of preovulatory follicles. OT production by granulosa cells in culture was stimulated by the addition of hCG, but only in cultures derived from preovulatory follicles; whereas FSH was without any effect. Addition of extrinsic OT to the cultures elicited an increase in progesterone production only for the granulosa cells derived from preovulatory follicles. Together, the results of the in vivo and in vitro studies point to an involvement of OT in the process of leuteinisation in the marmoset monkey.

Animals↗

PubMed1994

Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes.

Experiments were conducted to develop a simple rapid-freezing protocol for mature mouse oocytes that would yield a high proportion of oocytes with developmental potential. The effects of concentration (3.5, 4.5 and 6.0 M dimethyl sulfoxide (DMSO) all with 0.5 M sucrose) and the duration of exposure (2.5 min vs 45 sec) of oocytes to the cryoprotectant and its extraction after thawing in 2, 3 or 4 steps of descending sucrose concentration were studied. The most effective of the rapid-freezing and thawing protocols (4.5 M DMSO; 45 sec exposure and 3-step thawing) was compared to slow freezing protocols using 1.5 M DMSO and 1.0 M 1,2 propanediol as cryoprotectants. The DMSO concentrations had an effect on survival, fertilization and embryo development using short (45 sec) but not long (2.5 min) exposure. The rate of morphological oocyte survival was significantly higher using 4.5 M DMSO than 3.5 or 6.0 M (92% vs 82 and 73%, respectively). The development of fertilized embryos to blastocysts was also significantly higher at 4.5 M than at 3.5 or 6.0 M (68% vs 42 and 53%, respectively). The extraction of cryoprotectant in 3 or 4 steps of descending sucrose concentration resulted in higher survival (P < 0.01) and fertilization than in 2 steps. The best survival, fertilization and development was achieved with the 3-step procedure. Optimal combinations of conditions were 4.5 M DMSO at 45 sec prefreeze exposure and 3-step extraction of the cryoprotectant. Oocytes frozen by conventional methods had a survival, fertilization and development to blastocyst rate significantly lower than those frozen under the optimal rapid conditions. Thus rapid freezing of mature mouse oocytes with 4.5 M DMSO + 0.5 M sucrose and short prefreeze exposure is effective and has the additional advantage of being less time-consuming than slow freezing methods.

Journal Article↗

PubMed1994

Oxytocin gene expression and oxytocin immunoactivity in the ovary of the common marmoset monkey (Callithrix jacchus).

Oxytocin was identified in ovaries recovered on Day 5 (+/- 1) of the luteal phase from three female marmoset monkeys (Callithrix jacchus). With use of a reverse transcription-polymerase chain reaction assay, expression of mRNA for oxytocin and oxytocin receptor was detected in both luteal tissue and in the ovarian remnant. Evidence for ovarian synthesis of oxytocin was provided by immunohistochemistry, which showed positive staining for oxytocin and neurophysin in the cytoplasm of the luteal cells. Some luteal cells had a more intensely stained perinuclear region than others for oxytocin immunoreactivity, whereas the staining for neurophysin was evenly distributed. Granulosa and theca cells of antral follicles also showed positive staining for oxytocin immunoreactivity; no reactivity was found in fibroblast or endothelial cells. Oxytocin immunoreactivity was also detected in the luteal tissue of all animals by immunoassay, with values ranging from 2.8 to 12.1 ng/g wet weight. The oxytocin concentration for the ovarian remnant was either very low (0.55-0.75 ng/g wet weight) or nondetectable (< 0.5 ng/g wet weight). Local production of oxytocin within the ovary was suggested by the measurement of higher oxytocin concentrations in the blood from ovaries containing corpora lutea compared with peripheral blood. Collectively, these results provide evidence for ovarian biosynthesis of oxytocin and suggest the possibility of a paracrine role in the regulation of primate ovarian function.

Animals↗

PubMed1994

Progestin content and biosynthetic potential of the corpus luteum of the African elephant (Loxodonta africana).

The aim of this study was to examine the progestin content and biosynthetic potential of the corpus luteum of the African elephant (Loxodonta africana). Luteal tissue was collected from nonpregnant and early, mid- and late pregnant elephants (n = 2 per group) shot in the Kruger National Park. Pieces of individual corpora lutea (2-3 per animal; 23 in total) were stored directly in ethanol before hormone analysis. Matching tissue pieces were incubated for 2 h with [3H]pregnenolone (2 x 10(5) c.p.m.), after which tissue plus medium were also stored in ethanol. Progesterone and 17 alpha-hydroxyprogesterone immunoreactivity in tissue extracts were determined by enzymeimmunoassay and radioimmunoassay, respectively, before and after reverse phase HPLC. Progesterone immunoreactivity predominated over that of 17 alpha-hydroxyprogesterone in all corpora lutea examined but concentrations of both hormones were very low (73-374 ng g-1 and 3-93 ng g-1, respectively after HPLC). There were no obvious differences in hormone concentrations in corpora lutea from animals at different reproductive stages. Progesterone and 17 alpha-hydroxyprogesterone immunoreactivity assayed before HPLC was 10-30 times higher than that measured after chromatographic separation. HPLC consistently revealed two large immunoreactive peaks associated with relatively nonpolar compounds, which together accounted for most (at least 75%) of all progesterone immunoreactivity measured. Large amounts of radioactivity with the same retention times as these peaks were also detected after HPLC in samples incubated with [3H]pregnenolone. Analysis of conversion products from four corpus luteum incubations indicated that between 52% and 84% of [3H]pregnenolone had been converted; 19-33% was accounted for by progesterone, and 12-50% by the two substances represented by the unidentified peaks. Subsequent GCMS analysis identified the two immunoreactive peaks as 5 alpha-pregnane-3 alpha-ol-20-one and 5 alpha-pregnane-3,20-dione (5 alpha-dihydroprogesterone). These results indicate that the major progestins contained within and biosynthesized by corpora lutea of African elephants are 5 alpha-reduced metabolites, and that progesterone and 17 alpha-hydroxyprogesterone are quantitatively of minor importance.

17-alpha-Hydroxyprogesterone↗

PubMed1994

Molecular mass and isoelectric properties of pituitary and urinary gonadotrophins in callitrichid primates.

In this study, the molecular masses and isoelectric characteristics of pituitary LH and FSH in two species of callitrichid primate, the common marmoset (Callithrix jacchus) and the cotton-top tamarin (Saguinus oedipus), were determined. Comparative data for urine samples from Callithrix jacchus are also presented. The separation of gonadotrophins from pituitary extracts and urine was performed under nonreducing conditions using SDS-PAGE and isoelectric focusing procedures. Hormone activity in gel eluates was determined by in vitro bioassays for LH and FSH and by a microtitre plate enzymeimmunoassay for LH. The molecular masses of pituitary and urinary proteins were between 36 and 37 kDa for LH and FSH, and were similar in both species. A dimer form of pituitary LH with a molecular mass of 33 kDa was also found in the cotton-top tamarin, but not in the marmoset. Guanidine-HCl dissociation of gonadotrophins from marmoset and tamarin pituitaries before electrophoresis gave proteins of 16 and 28 kDa, and 16 and 25 kDa range, respectively. Isoelectric focusing revealed numerous peaks of bioactivity for both LH and FSH, indicating the presence of multiple molecular variants (isoforms) of each hormone. In both species pituitary FSH eluted over a narrower and more acidic pH range than LH. Isoelectric focusing profiles for pituitary and urinary LH in the marmoset were similar (pH range 5.0-8.5), whereas urinary FSH demonstrated a more acidic profile than the pituitary protein. These results give comparative information on the properties of New World primate gonadotrophins, which should be useful in studies of their physiological action and in aiding the development of improved reagents and assays for their detection.

Animals↗