Showing papers in "Journal of Reproduction and Development in 1992"

Radioimmunoassay of Corticosterone using 125I-Labeled Radio-ligand

Abstract: A radioimmunoassay (RIA) of corticosterone was developed and characterized using 125I labeled radioligand (ICN Biomedicals, USA) and a single ether extraction procedure The intra- and interassay coefficients of variation was 72% and 146% and assay sensitivity was 164±35 pg per tube Utilizing this assay, the antiserum for corticosterone (GDN377) made small samples (01-10μl) use possible The ease of this technique allows handling of large numbers of samples as compared with RIA using 3H-corticosteroneThe specific steps in the procedure of assay were following: 125I-corticosterone was diluted with 005M phosphate buffered saline (PBS) containing 1 % bovine serum albumin (BSA) Antiserum (GDN377) was diluted with 005M PBS containing 005mM ethylenediamine tetraacetic acid, 2Na salt, dihydrate (EDTA) and 04% normal rabbit serum (NRS) to 1 : 15000-1 : 20000 dilution Goat anti-rabbit gamma globulin serum (ARGG) was diluted with 005M PBS containing 35% polyethylene glycol 6000 (PEG) to 1:150 dilution Plasma or serum (01-1μl) and standard were diluted to 400μl with distilled water in 13 × 100mm culture tubes and extracted once with 2ml of diethyl ether The samples were allowed to settle for 5 min to ensure separation of ether and aqueous phases The tubes were then placed in dry ice-ethanol bath to freeze the aqueous phase The ether phase was then decanted into 12 × 75mm culture tube and dried under reduced pressure with gentle mixing at 50C One hundred μl of PBS containing 1% BSA, antiserum and labelled ligand (50-100Bq/100μ) were added to each tube and the extract was dissolved by agitation and incubated at 4C for 24 h Then, 100μl of ARGG was added to each tube After further incubation for 24 h, the antibody bound hormone was separated from the free hormone by centrifugation at 4C 1700g for 30 min The supernatant was carefully decanted and the precipitate was counted in automatic gammacounter Plasma or serum concentrations of corticosterone were calculated using a standard curve

In Vitro Capacitation of Canine Spermatozoa

Abstract: Ejaculated canine spermatozoa were incubated at a concentration of 0.6-4.0×108/ml, using a modified Krebs-Ringer bicarbonate solution (TYH) at 37C under 5% CO2 in air. Motility, aggregation, viability, hyperactivated movement and acrosome reaction of spermatozoa were examined at 0, 1, 2, 3, 4, 5 and 6 h of incubation. Motility and viability decreased slightly and aggregation increased at 1h of incubation, then did not significantly change until 6h of incubation (about 3.4, 65% and 75%, respectively). The percentage of spermatozoa showed hyperactivated movement and acrosome reaction increased remarkably at 3 h (53.9%) and 4h (68.0%) of incubation, respectively. The 4 h-incubated spermatozoa were inseminated at a concentration of 0.5 or 1.0×106/ml to the oocytes incubated in TYH (bovine serum albumin (BSA) -) with 10% fetal bovine serum for 48 h or 72 h. The spermatozoa penetrated into the zona pellucida at 1h after insemination. At 2 h after insemination, the spermatozoa penetrated into the cytoplasm (34.0%), then the percentage increased significantly to 63.4% at 4h after insemination. These results suggest that ejaculated canine spermatozoa are capacitated by 4h incubation in TYH.

Immunohistochemical study on gonadotropin-releasing hormone neurons in the Shiba goat brain

Abstract: Gonadotropin-releasing hormone (GnRH) neurons were examined immunohistochemical-ly in the brain of mature female Shiba goats with a monoclonal antibody to GnRH (LRH-13, Park and Wakabayashi, Endocrinol.Jpn. 33: 257-272, 1986). In all animals, GnRH immunoreactive neurons exhibited a complex morphology consisting of round (49.6%), multipolar (39.8%) or fusiform (10.6%) cell bodies and fibers with beaded varicosities. GnRH cell bodies were distributed rostrally from the diagonal band of Broca and medial septum, passing through the medial and lateral preoptic areas, and caudally ending within the ventromedial anterior hypothalamic areas. A majority (62.6%) of GnRH cells were found in the preoptic areas adjacent to the organum vasculosum of the lamina terminalis, and relatively few cells (7.1%) were distributed in the arcuate nucleus or its vicinity. Preoptic GnRH neurons project their fibers to the tubero-infundibular sulcus of the median eminence by at least 2 routes: (1) the majority of fibers were observed in the periventricular area and the arcuate neuclus (periventricular pathway), and (2) less prominent ones were found in the anterior and ventrolateral hypothalamus (ventrolateral pathway). In addition to these hypothalamic regions, GnRH immunoreac-tive fibers were also found in the neurohypophysis.

Chromosomal Analysis of First-Cleavage Mouse Eggs Fertilized in Caffeine-Containing Medium

Abstract: Eggs derived from superovulated F1(BALB/c × C57BL/6) mice were fertilized in a modified Krebs-Ringer bicarbonate medium (TYH) containing 2-mM caffeine. Spermatozoa obtained from the cauda epididymis of ICR male mice were preincubated in the caffeine-containing for 2 h before the mixing of gametes. Gametes preincubated and inseminated in the caffeine-free medium served as controls. Chromosome analysis at the first cleavage showed a significantly (P<0.05) higher incidence of triploidy in eggs fertilized in the caffeine-containing medium (22.9%) than in the control (15.8%). The incidence of aneuploidy and the sex ratio were not significantly different between the two groups of eggs. Most of the triploid eggs observed were considered to be dispermic in origin.

Chemical castration by a single intratesticular injection of lactic acid in rats and dogs

Abstract: Chemical castration by a single intratesticular injection of lactic acid, which is used for cattle, was studied in rats and dogs. The injection caused necrosis of the testicular tissue in both species. Lesions in the testes were observed as early as 24 h after the injection. Plasma testosterone levels declined rapidly after treatment and remained low thereafter. The results showed that a long-lasting and probably irreversible suppression of spermatogenesis can be brought about easily and immediately by an injection of lactic acid into the testes, and that the procedure could be used for the castration of dogs.

Histochemical Demonstration of Activin/Inhibin .BETA.A-, .BETA.B- and .ALPHA.-Subunits in Early Embryos and Oviducts of Different Strains of Mice.

Abstract: Spontaneous arrest of cleavage takes place at the 2-cell stage (the 2-cell block) in vitro, in some mouse strains (the "blocking strains"), whereas in others, no such arrest has been recorded (the "non-blocking strains"). We found that activin A releases the 2-cell block, and stimulates the early embryogenesis in mice of the blocking strains (Lu et al. 1990). In this paper, presence of activin/inhibin subunits in zygotes, 2-cell embryos, and oviductal epithelial cells was demonstrated immunohistoche-mically, using the blocking (CD-1 and DBA/2NJcl) and the non-blocking (C57BL/6NCrj) strains of mice. Strong immunoreactivity for the βA-, and βB-subunits of activins/inhibins was detected, in the cytoplasm of 1-cell and 2-cell embryos of the 3 strains. Staining intensities with the βA-subunit was higher during the 2-cell stage than during the 1-cell stage in the blocking strains. In the non-blocking strains, no such increase was observed. In contrast with the case of βA-subunit, immunoreactivity of the embryos to the βB-subunit decreased in the blocking strains, while in the non-blocking strain, it stayed at the same level between the two stages. Possible role played by activin AB in the early embryogenesis, was discussed. The immunohistochemically detected activin/inhibin subunits in the zona, probably represented those transferred passively from the oviductal fluid.

Effects of Storage Conditions of Bovine Ovaries and Oocytes on the Success Rate of in Vitro Fertilization and Culture

Abstract: The present study was carried out to examine effects of storage conditions of bovine ovaries and/or oocytes on the success rate of in vitro fertilization (IVF) and subsequent in vitroculture (IVC) of the zygotes. Ovaries obtained from a slaughter house were stored in Ringer solution at 39C (group 1), 20C (group 2), or 4C (group 3). In another group, immature oocytes were isolated from ovaries and stored in TCM-199 medium at 39C (group 4). The entire collection procedures of the ovaries took approximately 3 h at the slaughter house from the time of slaughter. The ovaries and/or the oocytes were stored under the conditions described above for 5 h before the experiment. In the control group, oocytes were isolated from the ovaries and cultured immediately to induce maturation. Following IVF and IVC, none of the zygotes developed to morula stage in group 1 and 3. In group 2, some zygotes developed to morula or blastocyst stage (11%, 7% respectively), but the rates were significantly lower than in the control group (32%, 15%). Group 4 gave high success rates (38%, 13%) similar to those attained in the control gorup.

Effects of Prolonged Sperm Preincubation and Elevated Calcium Concentration on Fertilization of Cumulus-Free Mouse Eggs in Vitro

Abstract: Freshly superovulated eggs of Jcl: ICR mature mice that had been freed from their cumulus oophorus with hyaluronidase were fertilized by epididymal spermatozoa preincubated for various periods in a modified Krebs-Ringer bicarbonate medium (TYH). Calcium concentration in the fertilization medium was elevated to the twice (3.42 mM) or to the 3 times (5.13 mM) of TYH (1.71 mM) and all incubations were performed at 37C under 5% CO2 in air. Both the sperm preincubation time and the concentration of calcium in the fertilization medium were found to exert significant effects on fertilization of cumulus-free eggs in vitro, and the highest fertilization rate was obtained when the cumulus-free eggs were inseminated with 150 min-preincubated spermatozoa in a medium with 3.42 mM calcium.

Transplantation of Porcine Blastomere Nuclei into Oocytes Collected from Prepubertal Gilts.

Abstract: Feasibility of utilizing follicular oocytes derived from gonadotropin treated prepubertal gilts and matured in vitro in nuclear transplantation of porcine embryos as the recipient cytoplasts was examined. After culture of 806 oocytes collected from gilts which had received an injection of 1500 i.u. PMSG followed by 750 i.u. hCG 72 h later, 488 (60.5%) matured normally, whilst fewer oocytes matured (144/308, 46.8%, P<0.05) when gilts were treated with 2000 i.u. PMSG. The proportion of oocytes activated after electric stimulation by double pulses of 150 V/mm D.C. for 30 or 60, usec were 62.9% (22/35) and 73.2% (30/41), respectively. Of 433 matured oocytes 342 (79.0%) were successfully manipulated to remove about 1/3 of cytoplasm adjacent to the polar body, with 68 of 77 (88.3%) being confirmed after acridine orange staining to be successfully enucleated. Attempts of electrofusion of the enucleated oocytes with 6 to 8 cell stage blastomeres resulted in 82.5% (66/80) successful fusion. Sixteen (45.7%) of the 35 nuclear transplanted embryos cleaved to 2 to 4 cell stage in culture with modified Kreb's Ringer Bicarbonate solution supplemented with 10% sheep serum.

Sex Determination of Bovine Embryos by the Polymerase Chain Reaction Using Y-Specific Primers

Abstract: The sex of embryos was determined by the polymerase chain reaction (PCR) method using the DNA fragment of a sex-related gene or Y-chromosome specific nucleotide sequence By using the DNA fragment of the human sex determinant (SRY), cells of male humans, rabbits, rats, mice and goats can be distinguished from the female cells of each animal By PCR using DNA of the mouse sex determinant (Sry), male cells of mice and rats were selected from the cell population The sex of cattle cells and goat cells could be determined by PCR using BOV97M (cattle Y-specific repeated DNA sequence) The DNA preparation of rabbit or cattle embryos was amplified by the primer of either SRY or BOV97M, and the embryonic sex was successfully determined The sex of the embryos predicted using the H-Y antibody or sex chromosome was confirmed by PCR analysis for biopsy of the trophoblastic cells The bovine growth hormone gene (bGH) was used as an internal standard of cattle-specific DNA fragment High efficiency and accuracy for embryo sexing was attained from biopsied embryos Three of the 8 (38%) biopsied embryos transferred into the recipient resulted in pregnancy, and the sex of 2 calves born was the same as expected

Divergent Effects of Estrogens on the Somatotropic Axis in Male and Female Calves

Abstract: This study describes 24 h profiles of growth hormone (GH) and of cortisol in male and female prepubertal calves before, during and after treatment with estradiol-17β (E2, silastic implants, 45 mg). Plasma concentrations of estradiol, testosterone and of insulin-like growth factor-1 (IGF-1) were also recorded. No significant (P<0.05) sex differences in GH, IGF-1 and in cortisol secretion were observed in untreated animals before E2-treatment. GH means, baselines and peak amplitudes increased during the treatment and remained elevated 1 week after removal of E2 in female calves, but were unchanged in male calves. In contrast, IGF-1 plasma concentrations were increased in both sexes during and after E2-treatment. In males maximal IGF-1 concentrations were higher than in females. Cortisol profiles showed a high individual variability and no consistant changes related to sex or E2-stimulation. Based on the observation that the male calves already had a significant testosterone secretion, we conclude that the observed sex differences of GH-secretion in response to E2 are related to endogenous testosterone. The E2-stimulated increase of IGF-1 plasma concentrations in males appears as related to an altered GH responsiveness rather than to changes in GH secretory patterns.

Effects of Follicle Cells on in vitro Penetration of Pig Oocytes by Cryopreserved, Ejaculated Spermatozoa

Abstract: Cumulus-enclosed or denuded pig oocytes before and after maturation were inseminated with cryopreserved ejaculated spermatozoa in modified TCM-199. High proportions (79-94%) of cumulus-enclosed mature oocytes, but none of immature oocytes, were penetrated regardless of the presence of extra granulosa cells. However, significantly (P<0.001) higher penetration rates were always obtained in denuded mature and immature oocytes in the presence than in the absence of granulosa cells. All of the penetrated immature oocytes had decondensed sperm nuclei and remained at the germinal vesicle (GV) or condensed GV stage. The ability of granulosa cells to induce sperm capacitation and/or acrosome reaction was acquired fully when they were cultured for 36h at 39C. Frozen-thawed granulosa cell masses could support penetration of denuded mature oocytes in spite of complete loss in cell viability indicating that intercellular matrix and possibly cytosolic components liberated from damaged cells rather than components secreted from living cells may play an important role for inducing sperm capacitation and/or acrosome reaction and penetration of oocytes. The activity of the granulosa cell masses was completely lost by treatment of them at 60C for 30 min.

Bovine Early Pregnancy Factor: Purification and Biochemical Examination

Abstract: The isolation and purification of early pregnancy factor (EPF) in the serum of pregnant bovine is described. The serum of 10.4 1 was obtained from a pregnant bovine of 8 days after artificial insemination. The rosette inhibition titer (RIT) of the serum was 6. EPF was isolated using the diafiltration, ion-exchange chromatography (CM-Sepharose, DEAE-Sepharose) and FPLC-gel permeation chromatography. EPF active fraction was recognized in the elute (RIT=7) of 50 mM NaCl on the CM-Sepharose and the unadsorbed fraction (RIT ?? 8) on the DEAE-Sepharose. The unadsorbed fraction of DEAE-Sepharose had 4 bands by SDS-PAGE analysis. The molecular weight of 4 bands were 23, 22, 21.5, 21 KD respectively. Further, the fraction No. 32 of the FPLC had a high EPF activity (RIT ?? 8) and the molecular weight of this fraction was estimated as 2122 KD. The iso-electoric point of EPF was near 5.0 by 2D-SDS-PAGE analysis.

Changes in Two Kinds of 20α-Hydroxysteroid Dehydrogenase During the Luteal Phase in the Rat

Abstract: We have found previously that there are 2 kinds of 20α-hydroxysteroid dehydrogenase (20α-HSD) (EC 1.1.1.149) molecule (HSD1 and HSD2) in the mature rat ovary. In the present study, each enzyme activity was measured during pseudopregnancy (PSP) in the rat. Ovarian cytosol was collected in early (day 5), mid (day 9) or late (day 15) PSP. HSD1 activity was low in early and mid PSP and increased markedly at the end of PSP, while HSD2 activity was lower than HSD1 activity in early PSP and decreased as PSP advanced. These results suggest that the regulatory mechanism of the enzyme activity varies between HSD1 and HSD2, and that HSD1 mainly contributes to the increase in 20α-dihydroprogesterone secretion at the end of PSP. Interestingly, when ovaries at early and mid-term PSP were used, the total 20α-HSD activity in the cytosol fraction was increased markedly (163-296%) by passage through a DEAF column. This phenomenon was not observed with ovarian cytosol obtained at late PSP. Thus, we conclude that the ovary contains 2 different kinds of 20α-HSD molecule whose activities seem to be regulated separatedly, and that the HSD1 molecule is responsible for termination of the luteal phase.

The endogenous opioids do not mediate the suckling-induced suppression of LH secretion in lactating rats

Abstract: The effect of intravenous injection of naloxone on the suppression of LH secretion during lactation was examined in intact, ovariectomized, and ovariectomized steroid-primed lactating rats at early and late lactation. The day of parturition was designated day 0. The litter size was adjusted to 8 on day 1, and ovariectomy was performed on day 2. Progesterone alone or progesterone and estradiol in silicone capsules were implanted subcutaneously immediately after the ovariectomy. Blood samples were collected every 6 min for 3 h on day 8 and 16 in intact and ovariectomized rats and on day 16 in ovariectomized steroid-primed rats. Naloxone (total amount=1.2 mg) was injected intravenously via the sampling catheter every 6 min for 2 h after the first hour of the sampling period. Intravenous injection of naloxone had no significant effect on plasma concentrations of LH on day 8 in intact or ovariectomized lactating rats. On day 16, naloxone treatment increased the plasma level of LH in intact lactating rats but not in ovariectomized or ovariectomized steroid-primed lactating rats. The present study clearly indicates that the endogenous opioid peptides do not mediate the suppression of the LH release by suckling stimulus, since intravenous injection of naloxone did not increase the LH secretion at early lactation when the suckling stimulus is the dominant factor suppressing LH release.

Differential Production of Estradiol by Subpopulations of Porcine Granulosa Cells

Abstract: Investigation of regulation of estradiol production by porcine granulosa cells is hindered by lack of a culture system that consistently produces estradiol for extended periods. Granulosa cells that are characterized by their strong attachment to each other (TB-cells) were compared to cells that lack such attachments (WA-cells) for their responses to FSH, LH, and EGF in androstenedione supplemented medium. TB-cells were separated from WA-cells by filtration and were predominantly aggregates that had a smooth contour on one side of the aggregate. After enzymatic disruption, TB-cells had greater estradiol production than WA-cells when cultured initially, and they maintained this advantage through day 8 when stimulated with FSH or LH. Progesterone production was greater for TB- than WA-cells during initial culture, but by day 8 FSH and LH stimulated progesterone production was similar for these two subpopulations. EGF increased DNA concentrations and decreased production of estradiol and progesterone by similar amounts for each cell type. When TB-and WA-cells were cultured together, estradiol, but not progesterone, production was reduced disproportionately. This indicates that WA-cells or some component of these pools of cells interact with TB-cells to reduce estradiol production. We conclude that TB-cells are primarily from the mural layers within the follicle and are more differentiated at time of collection. The means through which TB-cells sustain FSH stimulated estradiol production in culture and why WA-cells fail to achieve this level of production are unknown.

Production of a Calf from a Nuclear Transfer Embryo Using in Vitro Matured Oocytes.

Abstract: This study was conducted to examine the possibility of nuclear transplantation using the bovine oocytes matured in vitro. Sixteen-cell stage embryos (fresh, frozen or embryos developed from the oocytes matured and fertilized in vitro) were designated to donor cells. The follicular oocytes matured in vitro were used as enucleated recipient oocytes. The enucleated oocytes were fused with a blastomere from donor embryos by electrofusion. Most recipient oocytes (325/417, 78%) fused with donor cells. The reconstituted eggs developed to 2-cell stage when cultured on a layer of cumulus cells (187, 58%). Out of the 325 reconstituted eggs, 35(11%) developed into morulae and 9 (3%) into blastocysts stage respectively. Eighteen morula or blastocyst stage embryos were transferred nonsurgically to 9 recipient cows into 1-3 embryos per recipient. Two recipients were confirmed pregnant. One of recipients produced a live offspring resulting from a fresh donor blastomere. The present study showed that in vitro matured oocytes can be used as recipient cytoplasm.

Effects of Microinjection of Glutathione on Male Pronucleus Formation in Porcine Oocytes Matured in Vitro

Abstract: Porcine follicular oocytes matured in a modified Krebs-Ringer solution were microinjected with glutathione (GSH) and their male pronucleus (mPN) formation ability was examined after in vitro fertilization. While viability of the oocytes injected with 140 pl vehicle solution (91%) was comparable with non-injected control (93%), microinjections of 35 pl and 140 pl of GSH solution (240 mM) decreased oocyte viability dose dependently (76% and 47%, respectively), showing a toxicity of the highly concentrated GSH solution. Male PN formation rate was low in the control (23%) and was not affected by injection of vehicle solution (17%). In contrast, a microinjection of both 35 and 140 pl GSH solution increased the ability significantly (52% and 53%, respectively). These rates were, however, significantly lower than the rate (70%) of those matured in porcine follicular fluid, which have been suggested to be comparable with in vivo matured oocytes. These results suggest that the low mPN formation ability in the porcine oocytes matured in vitro might be attributed in part to their low intracellular GSH concentration.

Studies on Cytogenetic and Morphological Assessment of Bovine Embryos Fertilized in Vitro

Abstract: Cytogenetic and morphological study was carried out to assess the quality of bovine embryos fertilized in vitro. The major results were summarized as follows. 1) An optimal condition for the chromosome preparation of bovine preimplantation embryos was established using 2-cell embryos fertilized in vitro. 2) Chromosomal anomalies were observed in 7.1-36.4% of 89 two- to 32-cell embryos and these anomalies were caused by abnormal fertilization, especially by polyspermy, and abnormal cleavage. 3) At blastocyst stage, the incidence (18.6%) of chromosomal anomalies in the inner cell mass (ICM) separated from trophectoderm cells by immunosurgery was significantly lower than that (44.2%) in whole blastocyst. 4) Anti-bovine spleen cells rabbit serum was prepared and the method to count the cell numbers of ICM and trophectoderm of bovine blastocysts separately was developed by a differential fluorochrome staining technique using this antiserum. 5) Using this technique, it was found that the cell-cell contacts of ICM cells in blastocysts derived from in vitro fertilization were tighter than those from in vitro fertilization followed by culture in vitro and in a rabbit oviduct. 6) The delineation of each blastomere of ICM cells in blastocysts derived from in vitro fertilization of in vitro matured oocytes was improved by the transfer of the embryos to a rabbit oviduct from the 4-cell stage. 7) The dead ICM cells were observed in ICM cells from survived blastocysts after freezing and thawing by three-step or one-step methods. These findings show that the low developmental ability of bovine embryos fertilized in vitro is caused by an abnormal fertilization and their low pregnancy rates may be caused by the reduced cell proliferation and the loose cell-cell contact of ICM cells.

Changes in Ratios of Phagocytotic Macrophages in the Splenic Adherent Cells during Estrous Cycle and Pseudopregnancy in Rats

Abstract: We investigated changes in the ratio of phagocytotic cells (rPC) in splenic adherent cell population which were harvested from rats at different estrous cycle stages or during pseudopregnan-cy rPC was assessed using the horse radish peroxidase intake as an index Either the highest or lowest ratio was observed on the proestrous or the metestrous day during the course of estrous cycle The highest ratio observed on the proestrous day decreased to 50% by ovariectomy on an antecedent diestrous day, and treatment with either 17β-estradiol or progesterone nullified this decrease A decrease in rPC observed between the proestrous and estrous days was blocked by a dopamine agonist, CB-154 (150μg/rat), treatment on the proestrous day, and a simultaneous PRL treatment (25 μg/rat) counteracted this effect The induction of pseudopregnancy by cervical stimulation on the proestrous day severely decreased rPC on 2 days later, and this decrease was completely inhibited by the treatment with CB-154 Thereafter rPC gradually increased toward the end of pseudopregnancy, but a high rPC observed at the end of pseudopregnancy (day 12) was significantly decreased by an ovariectomy one day before These results indicate that rPC changes during estrous cycle and pseudopregnancy, and that this changes is induced at least by ovarian steroids and PRL

Overcoming the Mouse 2-Cell Block by Co-Culture with Established Cell Lines.

Abstract: To overcome the mouse 2-cell block, embryos resulting from in vitro fertilization (IVF) were co-cultured with established cell lines, namely, CHO-K1 (derived from Chinese hamster ovary), CHUIU (derived from Chinese hamster lung), and HEPM (derived from human embryonic palatal mesenchyme). Differences of embryonal development were observed in the different established cell line co-cultures. CHO-K1 were the best feeder cells for overcoming the block, while the CHL/IU exhibited this ability to a much lesser degree. HEPM was not able to overcome the block. Observations of the structure of these established cell lines, revealed variations in the morphology and density of the feeder cells. A large number of villi were observed on the surfaces of CHO-K1 and CHL/IU cells, and the cell density of CHO-K1 was much higher than that of CHL/IU. On the other hand, the surfaces of HEPM cells exhibited no villi or other structures. These findings suggest that the morphology and density of the feeder cells play a role in overcoming the 2-cell block.

Studies of Mammalian Implantation In Vitro (I): Production of Laminin from Human Decidual Cell Line and Its Effects on Murine Trophoblastic Outgrowth

Abstract: TTK-1 cell line from human first trimester decidual tissue carried the surface markers such as HNK-1 (Leu 7) and HLA-DR. This cell line was strongly suspected of being human natural suppressor cell line of bone marrow or lymphoid tissue origin. In addition, the cells were found to express laminin on its cell surface, as examined by indirect immunofluorescence (IIF) test, and also secrete it into culture supernates as examined by enzyme linked immunoassay (EIA) and immunostaining. The addition of the supernates from this cell line into the murine embryo culture induced the trophoblastic outgrowth of embryos in vitro in the same manner as murine EHS sarcoma derived laminin did. This phenomenon was considered to be due to laminin in the UK-1 supernates because the effect was completely blocked by the addition of anti-laminin antibodies into the embryo culture.

Androstenedione and Testosterone Levels in Peripheral Plasma of Male Shiba Goats during Development

Abstract: Developmental changes in testicular size and plasma androgen levels in male Shiba goats were studied during 2-12 months of age. Scrotal circumference increased rapidly until 5 months of age (18.6±0.9 cm; mean±SEM). Plasma androstenedione and testosterone reached peak levels of 0.23-0.50 ng/ml and 5.47-9.69 ng/ml, respectively as testicular size increased, and then showed a tentative decline with nadirs of 0.01-0.04 ng/ml and 0.26-0.94 ng/ml, respectively after full growth in testicular size. The fluctuation of androstenedione was in parallel with that of testosterone but the androstenedione levels were only 1-10% those of testosterone. Thus, the results indicated that testosterone was the predominant androgen starting from 2-3 months of age through puberty in male Shiba goats.

Seasonal Changes in Androgen Release in Two Breeds of Goats with Different Seasonality

Abstract: Seasonal changes in peripheral plasma androgen levels in male goats were studied in 3 Saanen goats (seasonal breeders) and 3 Shiba goats (continuous breeders). Hourly blood sampling was carried out for 24 h in May (spring) and November (autumn) for measurement of plasma concentrations of testosterone and androstenedione. There were episodic bursts of secretion of androstenedione and testosterone during the 24 h periods in both May and November in both breeds. The mean levels of testosterone were 7.0-9.1 fold higher in November than those in May in the Saanen goat, whereas 1.2-2.9 fold higher in the Shiba goat. The degree of changes in the peak magnitude and the base-line level of testosterone in each individual in November were also somewhat smaller in the Shiba goat than in the Saanen goat. The plasma androstenedione levels in both breeds showed a similar change to testosterone, but the magnitude of fluctuation was only 1-10% of that in testosterone. These results suggest that the pattern of androgen release in male goats is episodic and follows marked seasonal changes, which are more conspicuous in Saanen breed.

Immunoreactive .BETA.A Subunit of Inhibin/Activin Is Present in Cytoplasm of Rat Spermatogenic Cells.

Abstract: The localization of βA subunit of inhibin/activin in rat testis was studied by means of immunohistochemistry using antibody raised against synthetic polypeptide. The intense reaction with anti-βA antibody was shown in the cytoplasm but not in the nucleus and the plasma membrane of spermatocytes. The reaction was also found in the cytoplasm of spermatids, through the immunostaining pattern was different between the two cells; the granule-like one in spermatocytes and diffused one in spermatids, respectively. Furthermore, the plasma membrane became positive in spermatids, suggesting that βA subunit may be produced in spermatocytes and secreted from spermatids. The expression of βA subunit in spermatogenic cells is thought to be stage-specific, because the reaction was not found in spermatogonia and spermatozoa. Although both Sertoli and Leydig cells are reported to produce βA subunit of inhibin/activin, no reaction was observed in these two cells. This suggests that the antibody used in this study may recognize the epitopes different from those detected by previously-used immunological probes.

Oocyte Membrane Components Preventing Trypsin-Induced Germinal Vesicle Breakdown in the Surf Clam Oocytes

Abstract: Maturation of surf clam oocytes can be induced with sperm, KCl, serotonin or calcium ionophore. We now report that the oocytes treated with trypsin can also induce germinal vesicle breakdown (GVBD). Oocytes incubated in 0.1% trypsin solution for 0.5 to 1 h, subsequently washed and incubated in control medium for 1 to 2 h underwent maturation. Treatment with glycosidase, DNase and RNase did not induce GVBD. The optimal condition for the induction of maturation was to incubate oocytes in 0.1 % trypsin solution for 1 h and to transfer the oocytes to a medium composed of 0.4 M Tris-HCl, pH 7.4, 20 mM CaCl2. The occurrence of spontaneous GVBD was dependent upon the presence of Ca2+ in the suspending medium. The optimal concentration of Ca2+ was 20 mM. To show that a membrane component is involved in sustaining maturation arrest, oocytes were incubated in a medium containing 1 M urea, 5 mM EDTA, 10 mM Tris-HCl, pH 7.4, for 30 min. The extracted membrane protein at final concentrations of 0.4 to 0.8 mg/ml blocked GVBD of trypsin treated oocytes. To identify the active component, the extracted membrane protein preparations were fractionated by gel filtration through a Sephadex G-100 column. The active principle had an estimated Mr of 18 kD based on elution position of standard proteins on gel filtration. The partially purified product was active at a concentration of 0.1-0.2 mg/ml. The present findings suggest that maturation arrest is sustained by an oocyte membrane protein and the resumption of oocyte maturation results from the hydrolysis of this substance(s) by a protease in the presence of Ca2+.

Effects of Season, Age at Calving, Time after Calving and Interval of Treatment on the Embryo Production in Superovulated Holstein Donors

Abstract: Effect of factors affecting the embryo production of superovulated Holstein cows were evaluated. Data were obtained from 455 superovulation-collections using 84 donors. For all of the hormonal treatment, FSH and PGF2α were used. Season (every 12 month), year (1988, 1989, 1990), age at calving (3, 4, 5, 6, ≥7 years old), time after calving (1st, 2nd, ≥3rd year), interval of treatment (1st treatment, 21-90, 91-150, ≥151 days) and interaction (season×year, age×time) were evaluated by analysis of variance for number of total ova/embryos (NT), number of fertilized ova (NF), number of normal embryos (NN) fertilization rate (%F) and normal rate (%N), and time after calving within each flush were evaluated by another model. The effects of season on NT, NF and NN, age at calving on NT, NF, %F and %N, and time after calving on NF, NN, %F and %N were significant (P<0.05), while effects of year, interval of treatment, season×year and age×time were not. Seasonal effects suggested the influence of heat. Effects of age at calving indicated a lag of peaks between NT (3 years) and %N (5 years), and no difference was observed among 3-6 years in NN. Influence of time after calving without the effect of repeated treatment was observed, and embryo production during the 3rd year or over was worse as compared with the 1st and 2nd year.