Female mice that are null for the FSH-receptor (FSH-R) gene are estrogen deficient, acyclic, and sterile. However, the heterozygous ( /−) mice initially have reduced fertility and stop breeding by 7–9 mo. The purpose of this study was to understand the basis of reduced fertility in mice with haploinsufficiency of the FSH-R. Heterozygous females were compared to / females at 3, 7, and 12 mo of age. By 7 mo most of the /− females were acyclic and <50% delivered pups. The wild-type females were normal in these respects. None of the 1-yr-old /− females gave viable offspring (73% in / ). Many degenerative changes, including atresia and apoptosis, and profound loss of oocytes, were apparent in /− mice by 7 mo. The 1-yr-old /− ovary had very few follicles and consisted mostly of fibroid tissue and cysts. Our data support the hypothesis that reproductive deficits in /− FSH-R mice occur because of accelerated oocyte loss due to increased cell death in the ovary. These events contribute to early reproductive senescence and biological aging in mice. Thus FSH-R status is an important determinant of ovarian aging and all phenomena that arise from subsequent estrogen deficiency and other aberrations.

Reproductive aging in female mammals is characterized by a progressive decline in fertility due to loss of follicles and reduced ovarian steroidogenesis. In this study we examined some of the endocrine and signaling parameters that might contribute to a decrease in ovulation and reproductive performance of mice with haploinsufficiency of the FSH receptor (FSH-R). For this purpose we compared ovarian changes and hormone levels in FSH-R heterozygous ( /−) and wild-type mice of different ages (3, 7, and 12 mo). Hormone-induced ovulations in immature and 3-mo-old /− mice were consistently lower. The number of corpora lutea (CL) were lower at 3 and 7 mo, and none were present in 1-yr-old /− females. The plasma steroid and gonadotropin levels exhibited changes associated with typical ovarian aging. Plasma FSH and LH levels were higher in 7-mo-old /− mice, but FSH levels continued to rise in both genotypes by 1 yr. Serum estradiol and progesterone were lower in /− mice at all ages, and testosterone was several-fold higher in 7-mo-old and 1-yr-old /− mice. Inhibin α (Western blot) appeared to be lower in /− ovaries at all ages. FSH-R (FSH* binding) declined steadily from 3 mo and reaching the lowest point at 1 yr. LH receptor (LH* binding) was high in the 1-yr-old ovary, and expression was localized in the stroma and interstitial cells. Our findings demonstrate that haploinsufficiency of the FSH-R gene could cause premature exhaustion of the gonadal reserves previously noted in these mice. This is accompanied by age-related changes in the hypothalamic-pituitary axis. As these features in our FSH-R /− mice resemble reproductive failure occurring in middle-age women, further studies in this model might provide useful insights into the mechanisms underlying ovarian aging.

It has been proposed that the antimitogenic action of progesterone (P₄) is mediated through a membrane receptor that has GABA_A receptor-like characteristics. To test this hypothesis, studies were designed to compare the antimitogenic effects of P₄ with its gamma amino butyric acid_A (GABA_A) receptor-activating metabolite, 5α-pregnane-3α–21-diol-20-one (5α3α). These studies revealed that P₄ was more effective than 5α3α in blocking mitogen-dependent mitosis of both small granulosa cells (GCs) and spontaneously immortalized granulosa cells (SIGCs). Ligand binding studies illustrated that P₄ bound to SIGCs with an apparent dissociation constant (K_d) of 0.32 ± 0.09 μM, whereas 5α3α bound with an apparent K_d of 40 ± 19 μM. Further, the GABA_A antagonist, bicuculline, did not attenuate P₄'s antimitotic action in SIGCs. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that none of the 6 known α chains of the GABA_A receptors to which bicuculline binds were detected in SIGCs. Taken together, these studies suggest that P₄ does not mediate its action via a GABA_A-like receptor. Additional studies revealed that P₄ regulated intracellular free calcium levels ([Ca² ]_i) as part of its antimitotic action. Specifically, P₄ maintained a basal [Ca² ]_i level that was slightly lower than normal. Increasing extracellular calcium not only increased basal [Ca² ]_i but also attenuated P₄'s antimitogenic effect. P₄'s actions appeared to be initiated at the membrane, since horseradish peroxidase conjugated-P₄ (HP-P₄), which is cell impermeable, was as effective in blocking mitosis as P₄. Progesterone receptor (PR) mRNA was not detected in SIGCs by RT-PCR analysis, which is consistent with the findings in GCs. However, a 60-kDa protein was detected within crude membrane fractions of both GCs and SIGCs using an antibody directed against the ligand binding domain of the PR (C-262). This antibody was also used in immunocytochemical studies to detect a protein that was associated with the plasma membrane of SIGCs. It is proposed that this 60-kDa protein mediates P₄'s membrane-initiated actions.

Haploid development is a normal part of the life cycle for some animals, but it has not been observed in mammals. Studies in mice have revealed that the preimplantation developmental potential of haploid embryos is significantly impaired relative to diploid embryos. The reasons for the severely limited developmental potential of haploid embryos in mammals have not been discerned. To examine the effects of haploid development on gene expression, and in particular on X-linked gene expression, and to evaluate to what degree newer techniques of producing and culturing such embryos might affect developmental potential, haploid and diploid parthenogenetic and androgenetic embryos were produced and reevaluated for developmental potential, genomic integrity, and relative expression levels of specific autosomal and X-linked gene transcripts. Our data confirm the previously observed restriction in haploid developmental potential, eliminate chromosomal abnormalities as a major factor in this restriction, and reveal subtle alterations in gene expression. Haploid parthenogenones display only very subtle alterations in the expression of most mRNAs but a consistent elevation in X-linked Bex1 mRNA expression. Haploid androgenones seem to lack repression of the Pgk1 gene that is seen in diploid androgenones, but this may reflect ongoing loss of those haploid androgenones that experience X chromosome inactivation. The significance and possible explanations for these differences are discussed.

The presence of prion protein in sperm and fluids collected from different parts of the ram genital tract was investigated by immunoblotting with monoclonal antibodies. A slightly immunoreactive 25- to 30-kDa protein was recognized on Western blots of testicular and epididymal sperm extracts. Immunoreactivity increased on ejaculated sperm extracts and 2 other bands at 35 and 43 kDa also reacted. Seminal plasma showed several immunoreactive bands, the main bands being detected at 43 and 35 kDa, whereas less reactive bands were observed at 30, 25, 20, and <14 kDa. All these bands strongly decreased in the seminal plasma after vasectomy, indicating a testicular or an epididymal origin. Testicular fluid showed almost no reactivity, whereas caudal epididymal fluid contained the 2 strong immunoreactive bands at 43 and 35 kDa and in some cases a faint 30-kDa band. The 43-kDa band was also found in the fluid from the proximal caput, whereas the 35-kDa band appeared in the distal caput. Immunoprecipitation of ³⁵S-labeled proteins secreted in the epididymal fluid indicated that the 43-kDa form was synthesized in caput and caudal regions and the 35-kDa form in the distal caput to the distal corpus. Treatment of caudal fluid and seminal plasma by N-glycosidase resulted in the formation of 3 bands: 1 highly reactive at about 25 kDa, a second less reactive at about 28 kDa, and a third at approximately 20 kDa. The pattern of prion protein distribution in epididymal fluids was found to be similar in scrapie-infected rams to that of healthy rams. Cauda epididymal fluid and seminal plasma from infected animals could not be treated directly with proteinase K, because of the presence of protease inhibitors. However, the prion protein immunoprecipitated from these fluids was completely cleaved by proteinase K, whereas in the same conditions this from an infected sheep brain gave the usual resistant band pattern.

The objectives of this study were to determine the effects of in vitro embryo production on histological development and gene expression in the skeletal muscle of bovine fetuses during late gestation. Blastocysts produced in vivo were obtained from superovulated Holstein cows. Blastocysts produced in vitro were obtained from oocytes of Holstein cows that were matured and fertilized in vitro. Single blastocysts were transferred into heifers at a synchronized estrous and fetuses were recovered at Day 222 of gestation (n = 12 each for in vivo and in vitro). Samples of semitendinosus muscle were obtained for histological analysis and assessment of gene expression. Individual muscle sections were stained for the assessment of primary muscle fibers, secondary muscle fibers, or total muscle fibers. Semiquantitative reverse transcription-polymerase chain reaction assays were performed for 5 different candidate genes. The ratio of secondary-to-primary fiber number was greater in fetuses from embryos produced in vitro compared with fetuses from embryos produced in vivo. Similarly, the ratio of secondary-to-primary fiber volume density tended to be greater in fetuses from embryos produced in vitro. The proportional volume of tissue present between myofibrils was greater in fetuses from embryos produced in vitro. The expression of mRNA for myostatin was decreased in skeletal muscle of fetuses in the in vitro group compared with controls. The expression of mRNA for glyceraldehyde-3-phosphate dehydrogenase tended to be increased in skeletal muscle of fetuses in the in vitro treatment group. There was no effect of treatment on the expression of mRNAs for myf-5, myoD, or myogenin. In conclusion, in vitro production of embryos resulted in fetuses with altered development of skeletal muscle fibers. Myostatin was identified as the candidate gene whose expression may contribute to the observed changes in muscle development of these fetuses.

Pronuclear formation, and the chromosomal constitution and developmental capacity of bovine zygotes formed by intracytoplasmic sperm injection with freeze-dried (lyophilized) spermatozoa were evaluated. Frozen-thawed spermatozoa were selected, freeze-dried, and stored at 4°C until use. After 22–24 h of in vitro maturation oocytes were denuded and injected singly with a lyophilized spermatozoon. Injected oocytes were activated by treatment with 10 μM ionomycin (5 min) alone and in combination with 1.9 mM 6-dimethylaminopurine (DMAP) for 4 h. Ionomycin plus DMAP activation treatment resulted in a significantly higher proportion of sperm-injected oocytes with two pronuclei than was found after activation with ionomycin alone (74% vs. 56%; P < 0.03). The rates of cleavage, morula, and blastocyst development of sperm-injected oocytes treated with ionomycin plus DMAP were higher than after activation with ionomycin alone (63.3%, 34.2%, and 29.6% vs. 44.7%, 18.7%, and 10.6%, respectively; P < 0.05). Seventy-three percent of blastocysts produced with lyophilized sperm were diploid. These results demonstrate that in vitro-matured bovine oocytes can be fertilized with freeze-dried sperm cells, and that resultant zygotes can develop into karyotypically normal blastocysts.

Propylthiouracil (PTU) is a thioamide drug used clinically to inhibit thyroid hormone production. However, PTU is associated with some side effects in different organs. In the present study, the acute and direct effects of PTU on testosterone production in rat Leydig cells were investigated. Leydig cells were isolated from rat testes, and an investigation was performed on the effects of PTU on basal and evoked-testosterone release, the functions of steroidogenic enzymes, including protein expression of cytochrome P450 side-chain cleavage enzyme (P450_scc) and mRNA expression of the steroidogenic acute regulatory protein (StAR). Rat Leydig cells were challenged with hCG, forskolin, and 8-bromo-cAMP to stimulate testosterone release. PTU inhibited both basal and evoked-testosterone release. To study the effects of PTU on steroidogenesis, steroidogenic precursor-stimulated testosterone release was examined. PTU inhibited pregnenolone production (i.e., it diminished the function of P450_scc in Leydig cells). In addition to inhibiting hormone secretion, PTU also regulated steroidogenesis by diminishing mRNA expression of StAR. These results suggest that PTU acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450_scc function and StAR expression.

Mammalian sperm motility, capacitation, and the acrosome reaction are regulated by signal transduction systems involving cAMP as a second messenger. Levels of cAMP are controlled by two key enzymes, adenylyl cyclase and phosphodiesterases (PDEs), the latter being involved in cAMP degradation. Calmodulin-dependent PDE (PDE1) and cAMP-specific PDE (PDE4) activities were previously identified in spermatozoa via the use of specific inhibitors. Here we report that human sperm PDEs are associated with the plasma membrane (50%–60%) as well as with the particulate fraction (30%–50%) and have more affinity for cAMP than cGMP. Immunocytochemical data indicated that PDE1A, a variant of PDE1, is localized on the equatorial segment of the sperm head as well as on the mid and principal pieces of the flagellum, and that PDE3A is found on the postacrosomal segment of the sperm head. Immunoblotting confirmed the presence of PDE1A and PDE3A isoforms in spermatozoa. Milrinone, a PDE3 inhibitor, increased intracellular levels of cAMP by about 15% but did not affect sperm functions, possibly because PDE3 represents only a small proportion of the sperm total PDE activity (10% and 25% in Triton X-100 soluble and particulate fractions, respectively). PDE1A activity in whole sperm extract or after partial purification by anion-exchange chromatography was not stimulated by calcium calmodulin. Results obtained with electrophoresis in native conditions indicated that calmodulin is tightly bound to PDE1A. Incubation with EGTA EDTA, trifluoperazine, or urea did not dissociate the PDE1A-calmodulin complex. These results suggest that PDE1A is permanently activated in human spermatozoa.

Ethylnitrosourea (ENU), a well known alkylating agent, induces congenital anomalies in fetuses when it is administered to pregnant animals. In previous studies, we reported that ENU induced apoptosis and growth arrest in fetal tissues and organs immediately after its administration to pregnant rats. In the present study, we investigated the histopathological changes of the placenta after ENU administration to pregnant rats on Day 13 of gestation (GD13) to obtain a clue for clarifying the role of the placenta in the process of fetal developmental disability induced by genotoxic stress. Apoptotic cells increased and DNA-replicating cells decreased in the trophoblastic cells in the placental labyrinth zone of the ENU-treated group by 3 h after treatment. The number of apoptotic cells peaked at 6 h after treatment and returned to control levels at 48 h after treatment. The number of DNA-replicating cells reached minimum levels at 6 h after treatment and returned to control levels at 48 h after treatment. By immunohistochemistry, p53-positive signals were observed in trophoblastic cells in the labyrinth zone of the ENU-treated group from 3 to 6 h after treatment. Significant decreases in fetal and placental weights were observed in the ENU-treated group at 2 days (GD15) and 8 days (GD21) after treatment. A reduction in the thickness of the labyrinth zone was histopathologically significant in the ENU-treated group. These results indicate that ENU induces apoptosis and growth arrest not only in fetal tissues, but also in trophoblastic cells in the rat placental labyrinth zone, and these placental changes may have roles in the induction of fetotoxicity and teratogenicity of ENU. Moreover, a possible involvement of p53 in the induction of apoptosis and growth arrest is suggested.

The purpose of the present studies was to examine the role and regulation of the antiapoptotic Flice-like inhibitory protein (FLIP) in rat granulosa cells by tumor necrosis factor α (TNFα) in vitro. Granulosa cells from immature rats primed with eCG were cultured in serum-free RPMI in the absence or presence of TNFα (20 ng/ml), cycloheximide (CHX, 10 μg/ml), SN50 (a specific inhibitor of nuclear factor κB [NFκB] translocation, 100 or 200 μg/ml), or a combination of these. (SM50, a mutated inactive peptide of SN50, was used as control.) Inhibitor κB (IκB; total and phosphorylated forms) and NFκB binding abilities were measured by Western blot and electrophoretic mobility shift assay, respectively. Apoptosis was assessed by in situ TUNEL assay, whereas FLIP mRNA levels were determined by semiquantitative reverse transcriptase-polymerase chain reaction. TNFα alone failed to induce granulosa cell death but significantly increased the apoptotic cell number in the presence of cycloheximide. TNFα significantly up-regulated the expression of the short form of FLIP (FLIP_S) but not the long form (FLIP_L). TNFα induced IκB phosphorylation and NFκB activation. SN50, but not SM50, attenuated TNFα-induced FLIP_S expression and enhanced TNFα-induced apoptosis. Down-regulation of TNFα-induced FLIP_S by FLIP_S antisense expression enhanced TNFα-induced apoptosis. A full length of rat FLIP_S, with high homology to mouse FLIP_S (85%), had been cloned and sequenced. These findings suggest that, in addition to its proapoptotic function, TNFα can induce an intracellular survival factor for the maintenance of follicular development. TNFα-induced, NFκB-mediated FLIP_S expression is a determinant of granulosa cell fate.

The present study demonstrated that brief treatment of in vitro-matured porcine oocytes with demecolcine results in a membrane protrusion that contains a condensed chromosome mass, which can be easily removed by aspiration. This simple, chemically assisted method for removing maternal chromosomes enabled the production of a large number of nuclear-transferred porcine eggs. The development of eggs whose chromosomes were removed by this procedure following transfer of somatic cell nuclei to the blastocyst stage was not significantly different among groups activated using different procedures (6% to 11%) and was also not different among donor cells of different origins (3% to 9%), except for cumulus cells (0.4%). After transfer of 180 to 341 nuclear-transferred eggs that received somatic cells to 6 recipients, 2 of the recipients produced 8 healthy cloned piglets from the heart cells of a female pig. The chemically assisted method for removing maternal chromosomes was also effective for bovine and rabbit eggs.

Successful embryonic development is dependant on time and location-specific expression of appropriate genes. Unfortunately, information on stage-specific gene expression during early embryonic development in the bovine is lacking. In the present study, we compared gene expression between in vitro-produced Day 7–8 intact blastocysts (driver) and Day 9–10 hatched blastocysts (tester) using suppression-subtractive hybridization. Pools of 30 embryos for both driver and tester were used in the RNA extraction process. From limited amounts of starting material (∼400 ng of total RNA), a reverse transcription-polymerase chain reaction (PCR) procedure was used to amplify the mRNA and generate sufficient cDNA to conduct suppression-subtractive hybridization. The subtracted cDNA products were cloned, and 126 cDNAs representing expressed mRNAs were isolated, sized, single-pass sequenced, and compared to known sequences in GenBank. Ninety-two clones provided sequence information for further analysis. Among these, 31 exhibited high homology to known genes. Three, 26S proteasomal ATPase (PSMC3), casein kinase 2 α subunit (CK2), and phosphoglycerate kinase (PGK) were selected and further characterized using real-time quantitative PCR to assess their differential expression in hatched blastocysts. Overall, a 1.3-, 1.6-, and 1.5-fold increase in expression level was observed in hatched blastocysts compared with intact blastocyst for PSMC3, CK2, and PGK, respectively. These results show that construction of subtracted cDNA libraries from small numbers of embryos is feasible and can provide information on gene expression patterns during preattachment embryogenesis.

Hepatocyte growth factor (HGF) down-modulates FSH-dependent estradiol-17β (E₂) production in ovarian granulosa cells in vitro. The mechanisms of action underlying the antiestrogenic effects of HGF are vague, although evidence indicates that HGF may affect cAMP signal transduction in rat granulosa cells. The present study investigated the effects of HGF on FSH-induced steroidogenesis in the presence and absence of insulin-like growth factor I (IGF-I), as well as the actions of HGF within cyclic nucleotide-dependent signal transduction cascades in granulosa cells. Immature rat granulosa cells were incubated with FSH, IGF-I, and HGF. HGF impaired the production of FSH-stimulated and FSH IGF-I-stimulated E₂ synthesis, as well as FSH IGF-I-dependent estrone production. Progesterone synthesis was not altered by HGF. HGF suppressed FSH-dependent cAMP content at 24 h, but not at 36 h; cGMP content was stimulated by HGF with and without FSH at 24 h. In the presence of the cyclic nucleotide phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX), FSH-dependent cAMP accumulation was not affected by HGF. The suppressive effect of HGF on FSH-dependent E₂ production was alleviated by IBMX, whereas the HGF-dependent block in FSH IGF-I-supported E₂ production was not prevented by IBMX. The effects of HGF on cyclic nucleotide PDE activities were manifested in a time-dependent and hormone-dependent manner. FSH-induced cAMP PDE was suppressed by HGF at 24 h but not at 36 h, whereas FSH-dependent cGMP PDE was impaired at 36 h, but not at 24 h. HGF prevented the IGF-I-dependent reduction in FSH-stimulated cAMP-PDE activity at 24 and 36 h, and lowered FSH IGF-I-stimulated cGMP-PDE activity at 36 h, concomitant with an HGF-dependent increase in cGMP content at 24 h. These data indicate that HGF affects cAMP-directed and cGMP-directed signaling pathways at multiple sites in granulosa cells. These HGF-dependent effects may provide insight for mechanisms of action whereby HGF reduces E₂ secretion by granulosa cells.

Leukemia inhibitory factor (LIF) enhances in vitro murine preimplantation development in a time- and dose-dependent fashion. Knockout experiments have demonstrated that endometrial LIF is essential for in vivo murine implantation. We assessed the impact of LIF and an anti-LIF polyclonal antibody (pab) on in vivo development and developed a novel and successful nonsurgical method of embryo transfer for this species, a transcervical blastocyst transfer technique. The objectives of this study were to evaluate the effects of LIF and the anti-LIF pab on 1) implantation, resorption, pregnancy, and viability rates and 2) the overall structural and skeletal development. Two-cell embryos were recovered from superovulated mated donors, cultured to the expanded blastocyst stage, and transferred transcervically into pseudopregnant recipients. Exposure to 5000 U/ml LIF resulted in significant increases in implantation, pregnancy, and viability rates compared with controls. A similar dose of pab produced overall inhibitory effects with a significant decrease in implantation rate. Paradoxically, lower pab doses resulted in significantly increased viability rates. Exposure to LIF had no effect on fetoplacental development. However, pab treatments had variable but significant negative effects on placental length, ossification of the exoccipital bone, and vertebral space width compared with controls. Exposure of murine blastocysts to LIF at the time of transcervical transfer resulted in pronounced positive effects on implantation and pregnancy rates without affecting fetal development. A similar pab dose dramatically reduced implantation and pregnancy rates; at high and low doses, pab produced deleterious effects on placental and skeletal development.

The onset and regulation of puberty is determined by functional development of the brain-pituitary-gonad (BPG) axis. Sex steroids produced in the gonads play an important role in the onset of puberty. Stress interferes with reproduction and the functioning of the BPG axis, and cortisol has frequently been indicated as a major factor mediating the suppressive effect of stress on reproduction. Prolonged elevated cortisol levels, implicated in stress adaptation, inhibited pubertal development in male common carp (Cyprinus carpio). Cortisol treatment caused a retardation of pubertal testis development and reduced the LH pituitary content and the salmon GnRHa-stimulated LH secretion in vitro. A reduced synthesis of androgens also was observed. These findings suggest that the cortisol-induced inhibition of testicular development and the maturation of pituitary gonadotrophs are mediated by an effect on testicular androgen secretion. In this study, we combined cortisol treatment with a replacement of the testicular steroid hormones (testosterone and 11-oxygenated androgens) to investigate the role of these steroids in the cortisol-induced suppression of pubertal development. The effect of cortisol on spermatogenesis was independent of 11-ketotestosterone, whereas the effect on the pituitary was an indirect one, involving the testicular secretion of testosterone.

Growth differentiation factor-9 (GDF-9) is a glycoprotein secreted by the oocyte that is capable of stimulating granulosa cell proliferation and inhibiting differentiation. GDF-9 is a member of the transforming growth factor β superfamily of ligands known to signal through type I and II serine/threonine kinase receptors. In the sequenced human genome, seven type I and six type II receptors have been identified. Based on phylogenetic and sequence analyses, we predicted that GDF-9 likely interacts with known type I and type II receptors. We obtained soluble chimeric proteins with the ectodomains of candidate receptors fused to the Fc portion of immunoglobin and tested their ability to act as functional antagonists. Addition of bone morphogenetic protein receptor type II (BMPRII) ectodomain was most effective in blocking GDF-9 stimulation of granulosa cell proliferation and GDF-9 suppression of FSH-stimulated progesterone production. In addition, the ectodomains of bone morphogenetic protein receptor type IA, bone morphogenetic protein receptor type IB, and activin receptor type IIA were partially effective in blocking GDF-9 action. Furthermore, the BMPRII ectodomain directly interacted with GDF-9 in a coprecipitation study demonstrating the role of the BMPRII ectodomain as a binding protein for GDF-9. To demonstrate the role of BMPRII in GDF-9 signaling in follicular cells, the expression of this protein was blocked in cultured granulosa cells using specific BMPRII antisense oligomers. Inhibition of BMPRII biosynthesis completely prevented the GDF-9 induction of granulosa cell thymidine incorporation. GDF-9 expression is essential for early follicle development, and the presence of the type II and type I receptors in the neonatal rat ovary was verified by reverse transcription polymerase chain reaction. These results demonstrate the important role of BMPRII in mediating GDF-9 action in granulosa cells from small antral follicles and indicate that the effects of GDF-9 might be transduced by binding to BMPRII and one or more type I receptors.

The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuprolide acetate (LA) on follicular development and apoptosis-related mechanisms in preovulatory ovarian follicles (POF) obtained from prepubertal eCG-treated rats. Serum progesterone and estradiol levels were measured, and a significant decrease in circulating estradiol levels was observed in the LA group, whereas serum progesterone levels remained unchanged. Ovarian histology revealed an inhibitory effect of LA treatment on the follicular development induced by eCG. After 48 h of LA treatment, the numbers of atretic and preantral follicles were increased as compared with controls, whereas the number of antral follicles had decreased. Cells undergoing DNA fragmentation were quantified by performing in situ 3′ end labeling of DNA with digoxygenin-dUTP on ovarian sections. LA treatment caused an increase in the percentage of apoptotic cells in preantral and antral follicles. DNA isolated from these POF incubated 24 h in serum-free medium exhibited the typical apoptotic DNA degradation pattern. Treatment of follicles with epidermal growth factor (EGF) suppressed the spontaneous onset of DNA fragmentation, and a similar effect was observed in LA follicles. POF obtained from LA-treated rats showed no changes in Bcl-2 or Bax protein levels. However, a reduction in the Bcl-xL:Bcl-xS ratio was observed, with a greater decrease in Bcl-xL compared with Bcl-xS during the incubation, suggesting a lower stability of the Bcl-xL isoform in the LA group. These results indicate that in vivo GnRH agonist treatment produces an increase in the apoptosis process in POF from eCG-treated rats, and this effect is reversed in vitro by EGF. This GnRH analogue also reduced the stability of the Bcl-xL protein, thus interfering with follicular development by an as yet unknown mechanism.

Abortions of nuclear transfer (NT) embryos are mainly due to insufficient placentation. We hypothesized that the primary cause might be the aberrant allocations of two different cell lineages of the blastocyst stage embryos, the inner cell mass (ICM) and the trophectoderm (TE) cells. The potential for development of NT embryos to blastocysts was similar to that for in vitro fertilized (IVF) embryos. No difference in the total cell number was detected between NT and IVF blastocysts, but both types of embryos had fewer total cells than did in vivo-derived embryos (P < 0.05). The NT blastocysts showed a higher ratio of ICM:total cells than did IVF or in vivo-derived embryos (P < 0.05). Individual blastocysts were assigned to four subgroups (I: <20%, II: 20–40%, III: 40–60%, IV: >60%) according to the ratio of ICM:total cells. Most NT blastocysts were placed in groups III and IV, whereas most IVF and in vivo-derived blastocysts were distributed in group II. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocations of NT embryos to the ICM and TE cells during early development.

In adults, circulating leptin concentrations are dependent on body fat content and on current nutritional status. However, the relationships among maternal nutrient intake, fetal adiposity, and circulating leptin concentrations before birth are unknown. We investigated the effects of an increase in nutrient intake in the pregnant ewe on fetal adiposity and plasma leptin concentrations during late gestation. Between 115 and 139–141 days gestation (term = 147 ± 3 days gestation), ewes were fed a diet calculated to provide either maintenance (control, n = 6) or ∼155% of maintenance requirements (well-fed, n = 8). The fetal fat depots (perirenal and interscapular) were dissected, and the relative proportion of unilocular and multilocular adipocytes in each depot was determined. Maternal plasma glucose and leptin concentrations were significantly increased in well-fed ewes. Fetal plasma glucose concentrations were also higher in the well-fed group (115–139 days gestation: control, 1.65 ± 0.14 mmol/L; well-fed, 2.00 ± 0.14 mmol/L; F = 5.76, P < 0.04). There was no effect of increasing maternal feed intake on total fat mass, the relative mass of unilocular fat, or fetal plasma leptin concentrations (115–139 days gestation: control, 5.2 ± 0.8 ng/ml; well-fed, 4.7 ± 0.7 ng/ml). However, in both the control and well-fed groups fetal plasma leptin concentrations (y) were positively correlated with the relative mass of unilocular fat (x): y = 1.51x 1.70; (R = 0.76, P < 0.01). Thus, fetal leptin may play a role as a signal of unilocular fat mass in the fetus when maternal nutrient intake is at or above maintenance requirements.

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 ± 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 ± 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 ± 0.001 μg/g tissue), increased to Day 15 (6.96 ± 0.86 μg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.

Several strategies have been described for the primary culture of human myometrial cells. However, primary cultures of myometrial cells have a limited life span, making continual tissue acquisition and cell isolation necessary. Recent studies have demonstrated that cell culture life span is related to chromosomal telomere length, and cellular senescence results from progressive telomere shortening and the lack of telomerase expression. Transfection of cells with expression vectors containing the human telomerase reverse transcriptase (hTERT) maintains telomere length and effectively gives normal cells an unlimited life span in culture. In addition, hTERT extends the life span of cultured cells far beyond normal senescence without causing neoplastic transformation. In the present study, we developed a cell line from hTERT-infected myometrial cells (hTERT-HM). Cells were isolated from myometrial tissue obtained from women undergoing hysterectomy, and retroviral infection was used to express the catalytic subunit of telomerase in myometrial cells. Cells expressing hTERT have been in continuous culture for >10 mo, whereas the control culture senesced after approximately 2 mo. Telomerase activity was monitored in cells with a polymerase chain reaction-based telomerase activity assay. Telomerase-expressing cells contained mRNA for α smooth muscle actin, smoothelin, oxytocin receptor, and estrogen receptor α, but the estrogen receptor β receptor was lost. Immunoblotting analysis identified the expression of calponin, caldesmon, α smooth muscle actin, and oxytocin receptor. Although estrogen receptor expression was below the level of detection with immunoblotting, transfection experiments performed with reporter constructs driven by estrogen response elements demonstrated estrogen responsiveness in the hTERT-HM. In addition, treatment of hTERT-HM with oxytocin caused a concentration-dependent increase in intracellular calcium levels, confirming the presence of functional oxytocin receptors. Myometrial cells immortalized with hTERT retained markers of differentiation that are observed in primary cultures of smooth muscle cells. The expression of various smooth muscle/myometrium cell markers suggests that these cells may be an appropriate model system to study certain aspects of human myometrial function.

Endocrine-disrupting chemicals, known to be present in the environment, have great potential for interfering with reproductive health in wildlife and humans. There is, however, little direct evidence that endocrine disruption has adversely affected fertility in any organism. In freshwater and estuarine fish species, for example, although a widespread incidence of intersex has been reported, it is not yet known if intersexuality influences reproductive success. The purpose of this study was, therefore, to determine gamete quality in wild intersex roach (Rutilus rutilus) by assessing sperm characteristics, fertilization success, and ability to produce viable offspring. The results clearly demonstrate that gamete production is reduced in intersex roach. A significantly lower proportion of moderately or severely feminized fish (17.4% and 33.3%, respectively) were able to release milt compared with normal male fish from contaminated rivers (in which 97.6% of the males were able to release milt), reference male fish (97.7%), or less severely feminized intersex fish (experiment 1: 85.8%, experiment 2: 97%). Intersex fish that did produce milt produced up to 50% less (in terms of volume per gram of testis weight) than did histologically normal male fish. Moreover, sperm motility (percentage of motile sperm and curvilinear velocity) and the ability of sperm to successfully fertilize eggs and produce viable offspring were all reduced in intersex fish compared with normal male fish. Male gamete quality (assessed using sperm motility, sperm density, and fertilization success) was negatively correlated with the degree of feminization in intersex fish (r = −0.603; P < 0.001) and was markedly reduced in severely feminized intersex fish by as much as 50% in terms of motility and 75% in terms of fertilization success when compared with either less severely feminized intersex fish or unaffected male fish. This is the first evidence documenting a relationship between the morphological effects (e.g., intersex) of endocrine disruption and the reproductive capabilities of any wild vertebrate. The results suggest that mixtures of endocrine-disrupting substances discharged into the aquatic environment could pose a threat to male reproductive health.

The mammalian epididymis secretes numerous proteins important for sperm maturation. Among these are proteins D and E, which belong to the CRISP family (cysteine-rich secretory proteins) and are the product of the Crisp-1 gene. These proteins have been the focus of a number of studies and have been implicated in sperm/egg fusion. Protein D and protein E have been purified to apparent homogeneity in several laboratories. Polyclonal antibodies raised against each protein typically cross-reacted with both proteins, suggesting that they were immunologically similar, if not identical. Our laboratory has previously reported the generation of a monoclonal antibody (mAb 4E9) that recognizes only protein E. Using mAb 4E9, the localization of protein E was shown to be domain specific on the sperm surface and there is processing of the protein in the fluid, with only the lowest molecular weight form associating with sperm. Subsequent purification and amino acid sequencing of protein D confirmed that proteins D and E are nearly identical and differ only by presence of the 4E9 epitope on protein E. Here we report the generation of antibodies to regions of amino acid sequence identity in proteins D and E. Using these antibodies, we demonstrate that protein D associates with the sperm head and that a portion of this protein may be proteolytically processed. In addition, we demonstrate that the proteolytic processing of protein E occurs in the carboxy terminal region of this protein. The data also suggest that a portion of protein D may also undergo processing, similar to that of protein E. Finally, we use these antibodies to demonstrate that proteins D and E are differentially expressed by the epididymal epithelium. Taken together, these data suggest that proteins D and E may have individual roles in sperm function.

The golden hamster is the mammalian species in which intracytoplasmic sperm injection (ICSI) was first tried to produce fertilized oocytes. Thus far, however, there are no reports of full-term development of hamster oocytes fertilized by ICSI. Here we report the birth of hamster offspring following ICSI. Keys to success were 1) performing ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under a microscope with a red light source and 2) injecting sperm heads without acrosomes. All oocytes injected with acrosome-intact sperm heads died within 3 h after injection, while those oocytes injected with acrosomeless sperm heads survived injection. Under illumination with red light in a dark room, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally (77%), cleaved (91%), and developed into morulae (49%). Of the 47 morulae transferred to five recipient females, nine (19%) developed to live offspring.

The present study was conducted to examine the utility of rapidly matured oocytes as recipients for production of porcine embryos reconstituted with adult skin fibroblasts and whether arrest of meiotic resumption of recipient oocytes at the germinal vesicle (GV) stage by dibutyryl cyclic AMP (dbcAMP) improves in vitro developmental rates after reconstruction. At 24 h of maturation in the medium, 36.3% of oocytes reached the metaphase II (MII) stage. At 30 h of maturation, the percentage (71.4%) of MII oocytes did not significantly differ from that (78.0%) at 42 h of maturation. When MII oocytes recovered at 24 h of maturation were used as recipients, 22/156 (14.1%) cloned embryos developing to the blastocyst stage was significantly (P < 0.05) higher than those of embryos reconstituted with oocytes collected at 30 h (5/168; 3.0%) and 42 h (13/217; 6.0%) of maturation. Culture of oocytes in medium containing 1 mM dbcAMP for 20 h maintained 72.9% in the GV stage, whereas only 15.0% of nontreated oocytes were in the GV stage (P < 0.05). The effect of dbcAMP was reversible. However, the treatment of recipient oocytes with dbcAMP did not affect the development of reconstructed embryos when compared with nontreated oocytes. These results indicate that rapidly matured oocytes are superior in their ability to support development of porcine reconstructed embryos; however, arrest of meiotic resumption of recipient oocytes at the GV stage by dbcAMP does not improve reconstructed embryo developmental rates.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this experiment, the changes in Plk1 expression were detected in mouse oocytes through Western blotting. The subcellular localization of Plk1 during oocyte meiotic maturation, fertilization, and early cleavage as well as after antibody microinjection or microtubule assembly disturbance was studied by confocal microscopy. The quantity of Plk1 protein remained stable during meiotic maturation and decreased gradually after fertilization. Plk1 was localized to the spindle poles of both meiotic and mitotic spindles at the early M phase and then translocated to the middle region. At anaphase and telophase, Plk1 was concentrated at the midbody of cytoplasmic cleavages. Plk1 was concentrated between the male and female pronuclei after fertilization. Plk1 disappeared at the spindle region when microtubule formation was inhibited by colchicine or staurosporine, while it was concentrated as several dots in the cytoplasm after taxol treatment. Plk1 antibody injection decreased the germinal vesicle breakdown rate and distorted MI spindle organization. Our results indicate that Plk1 is a pivotal regulator of microtubule organization during mouse oocyte meiosis, fertilization, and cleavage and that its functions may be regulated by other kinases, such as staurosporine-sensitive kinases.

This study examined the fate of donor mitochondrial DNA during preimplantation development after nuclear transfer (NT) in cattle. Frozen-thawed cumulus cells were used as donor cells in the nuclear transfer. Mitochondrial DNA heteroplasmy in the nuclear transfer embryos was analyzed by allele-specific PCR (AS-PCR), direct DNA sequencing, and DNA chromatography. AS-PCR analysis for the detection of donor mitochondrial DNA was performed at the 1-, 2-, 4-, 8-, 16-cell, morula, and blastocyst stages of the embryos. The mitochondrial DNA from donor cells was detected at all developmental stages of the nuclear transfer embryos. However, mitochondrial DNA heteroplasmy was not observed in direct DNA sequencing of displacement-loop sequence from nuclear-transfer-derived blastocyst embryos. To confirm the mtDNA heteroplasmy in cloned embryos, the AS-PCR product from NT-derived blastocysts was analyzed by DNA sequencing and DNA chromatography. The nucleotides of NT-derived blastocysts were in accordance with the nucleotides from donor cells. These results indicate that the foreign cytoplasmic genome from donor cells was not destroyed by cytoplasmic events during preimplantation development that followed nuclear transfer.

We investigated the use of direct nuclear injection using the Piezo drill and activation by injection of stallion sperm cytosolic extract for production of cloned equine embryos. When metaphase II horse oocytes were injected with either of two dosages of sperm extract and cultured 20 h, similar activation rates (88% vs. 90%) and cleavage rates (49% vs. 46%) were obtained. The successful reconstruction rate of horse oocytes with horse somatic cell donor nuclei after direct injection using the Piezo drill was 82%. Four dosages of sperm extract (containing 59, 176, 293, or 1375 μg/ml protein) and two activation times (1.5–2 vs. 8–10 h after nuclear transfer) were examined. Cleavage and activation (pseudopronucleus formation) rates of oocytes injected with sperm extract containing 59 μg/ml protein were significantly (P < 0.05) lower than any other dosage. The percentage of embryos cleaving with normal nuclei in oocytes injected with the 1375 μg/ml preparation 1.5–2 h after donor injection was significantly (P < 0.05) higher than that of the 293 μg/ml preparation 8–10 h after donor injection (22 vs. 6%). Embryos developed to a maximum of 10 nuclei. Interspecies nuclear transfer was performed by direct injection of horse nuclei into enucleated bovine oocytes, followed by chemical activation. This resulted in 81% reconstruction (successful injection of the donor cell), 88% cleavage, and 73% cleavage with normal nuclei. These results indicate that direct nuclear injection using the Piezo drill is an efficient method for nuclear transfer in horse and cattle oocytes and that sperm extract can efficiently activate horse oocytes both parthenogenetically and after nuclear transfer

Cyclin E, a G₁ cyclin serving to activate cyclin-dependent kinase 2, is the only cyclin gene for which alternative splicing leading to structurally different proteins has been described. Different cyclin E proteins are present in tumor tissues but absent from normal (steady) tissues. Cyclin E contributes to the regulation of cell proliferation and ongoing differentiation and aging. Because trophoblast has invasive properties and differentiates into syncytium and placental aging may develop at term, we examined cyclin E protein variants in human placenta. Placental samples were collected from 27 deliveries between 33 and 41 wk and were compared with ovarian cancer (positive control). Both placental and tumor tissues showed seven cyclin E low molecular weight (LMW) bands migrating between 50 and 36 kDa. Placental expression of cyclin E showed certain variability among cases. Lowest cyclin E expression was detected in normal placentas (strong expression of Thy-1 differentiation protein in villous core and low dilatation of villous blood sinusoids). Abnormal placentas (significant depletion of Thy-1 and more or less pronounced dilatation of sinusoids) showed significant increase either of all (early stages of placental aging) or only certain cyclin E proteins (advanced aging). Our studies indicate that a similar spectrum of cyclin E protein variants is expressed in the placental and tumor tissues. Low cyclin E expression in normal placentas suggests a steady state. Overexpression of all cyclin E proteins may indicate an activation of cellular proliferation and differentiation to compensate for developing placental insufficiency. However, an enhanced expression of some cyclin E LMW proteins only might reflect an association of cyclin E isoforms with placental aging or an inefficient placental adaptation.

Early preantral mouse follicles with a diameter of 110–160 μm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.

Bioeffects after exposure to ultrasound are correlated to its duration. Although diagnostic ultrasound has been suggested to induce apoptosis, the underlying signal transduction pathway remains elusive. In this study, women in the first trimester of pregnancy were exposed to transvaginal diagnostic ultrasound with 5.0-MHz frequency for 0, 10, 20, or 30 min. The chorionic villi were obtained 4 h after exposure and activation of caspase-3 and cytochrome c release were analyzed by Western blotting. In contrast with the 0- and 10-min groups, cleavage products of active caspase-3 and cytochrome c release significantly increased in 20- and 30-min groups in a time-dependent manner. We show that long-duration exposure to transvaginal ultrasound activates effector caspase-3-mediated apoptotic cascade of chorionic villi in the first trimester of pregnancy. This occurs through the intrinsic death pathway involved in cytochrome c release. Our findings provide a molecular rationale for discriminant use of transvaginal ultrasound at the early stage of pregnancy.

Functional development of the adrenal cortex is critical for fetal maturation and postnatal survival. In the present study, we have determined the developmental profile of expression of the mRNA and protein of an essential cholesterol-transporting protein, steroidogenic acute regulatory protein (StAR), in the adrenal of the sheep fetus. We have also investigated the effect of placental restriction (PR) on the expression of StAR mRNA and protein in the growth-restricted fetus. Adrenal glands were collected from fetal sheep at 82–91 days (n = 10), 125–133 days (n = 10), and 140–144 days (n = 9) and from PR fetuses at 141–145 days gestation (n = 9) (term = 147 ± 3 days gestation). The adrenal StAR mRNA:18S rRNA increased (P < 0.05) between 125 days (7.44 ± 1.61) and 141–144 days gestation (13.76 ± 1.88). There was also a 13-fold increase (P < 0.05) in the amount of adrenal StAR protein between 133 and 144 days gestation in these fetuses. However, the amount of StAR protein (6.9 ± 1.7 arbitrary densitometric units [AU]/μg adrenal protein) in the adrenal of the growth-restricted fetal sheep was significantly reduced, when compared with the expression of StAR protein (17.1 ± 1.9 AU/μg adrenal protein) in adrenals from the age-matched control group. In summary, there is a developmental increase in the expression of StAR mRNA and protein in the fetal sheep adrenal during the prepartum period when adrenal growth and steroidogenesis is dependent on ACTH stimulation. We have found that, while the level of expression of StAR protein is decreased in the adrenal gland of the growth-restricted fetus during late gestation, this does not impair adrenal steroidogenesis. Our data also suggest that the stimulation of adrenal growth and steroidogenesis in the growth-restricted fetus may not be ACTH dependent.

Possible circadian fluctuations and long-term changes in concentrations of reproductive hormones in peripubertal female birds is poorly documented in comparison with mammalian species. Our objective was to document changes in concentrations of several reproductive hormones the several days before and after initial pubertal preovulatory surges of LH in turkey hens photostimulated with either constant (24L:0D) or diurnal (14L:10D) lighting. The hens were cannulated for hourly blood sampling, starting 10 days after photostimulation and continuing until all hens had laid at least two eggs. First eggs were oviposited between 16 and 24 days after photostimulation, and egg production ranged from two to nine eggs/hen during the experimental period. With both lighting treatments, concentrations of LH declined slightly, concentrations of progesterone (P₄) increased, and concentrations of estradiol-17β (E₂) were constant the 3–4 days prior to initial LH surges with no circadian fluctuations in hormone concentrations. Most (10 of 13) initial preovulatory surges of LH were coupled with ovulations, and all LH surges were coupled with P₄ surges. Those LH and P₄ surges not coupled with ovulations (blind surges) occurred with both lighting treatments, but the incidence of blind surges was higher with diurnal lighting. The interval between LH and P₄ surges was longer between the first and second surges than between subsequent surges, when the interval was approximately 26 h. The duration of LH surges (7.4 ± 3.0 h) was shorter than that of P₄ surges (10.0 ± 2.0 h). We conclude that, in the peripubertal female turkey, 1) prior to puberty (first LH-P₄ surges), there are no circadian fluctuations in concentrations of LH, P₄, and E₂, 2) 3 days prior to initial LH surges, E₂ concentrations are stable, LH concentrations decline slightly, and P₄ concentrations increase, and 3) surges of LH are coupled to surges of P₄ but LH-P₄ surges are not always coupled to ovipositions (blind surges), possibly because of internal ovulations.

α-Tocopherol transfer protein (α-TTP) was first described to play a major role in maintaining α-tocopherol levels in plasma, while α-tocopherol was primarily reported to be a factor relevant for reproduction. Expression of α-TTP is not only seen in the liver, from where it was first isolated, but also in mouse uterus, depending on its state of pregnancy, stressing the importance of α-TTP for embryogenesis and fetal development. The cellular localization of α-TTP in mouse uterus is reported here. By immunohistochemistry, α-TTP could be localized in the secretory columnar epithelial cells of the pregnant uterus on Days 4.5 and 6.5 postcoitum as well as in the glandular epithelial cells and the inner decidual reaction zone surrounding the implantation site. On Days 8.5 and 10.5 postcoitum (midterm of mouse pregnancy), α-TTP could still be detected in the uterine secretory columnar epithelial cells, while in α-TTP knockout mice, no immunostaining was visible. It is suggested that α-TTP plays a major role in supplying the placenta and consecutively the fetus with α-tocopherol throughout pregnancy. We conclude that α-tocopherol plays a role in the process of implantation and that α-TTP may be necessary for adequate α-tocopherol status of the fetus.

The presence of cAMP-dependent protein kinase (PKA) in the plasma membrane compartment and its association with an A-kinase anchoring protein (AKAP150) is implicated in mediating cAMP regulatory events in the rat myometrium. The association of PKA with purified myometrial plasma membrane declined gradually between Day 16 and Day 21 of gestation, with a decrease of 53% ± 11% of the catalytic subunit and of 61% ± 7% of the regulatory subunit at Day 21 compared with Day 19. To determine the role of progesterone in this association, pregnancy was prolonged by administration of progesterone or shortened by administration of the antiprogestin RU486. Progesterone treatment maintained PKA association with plasma membrane at Day 21 at 123% ± 23% (catalytic subunit) and 92% ± 4% (regulatory subunit) of Day 19 levels. In contrast, protein phosphatase 1, protein phosphatase 2B, phospholipase Cβ₃, and AKAP150 concentrations in the plasma membrane did not change over this interval or with progesterone treatment. Changes in PKA coimmunoprecipitated with membrane-associated AKAP150 paralleled those in total plasma membrane on Days 19 and 21 and on Day 21 following progesterone treatment. In contrast, plasma membrane PKA catalytic and regulatory subunits decreased by 20 h after RU486 injection on Day 15 of pregnancy to levels resembling those on Day 21. These data indicate that progesterone prevents the decline in PKA associated with myometrial plasma membrane and with AKAP150 in the pregnant rat. The decrease in membrane-bound PKA between Days 19 and 21 and after RU486 treatment precedes the onset of parturition in both experimental paradigms. The loss of plasma membrane PKA may be critical for the decrease in the inhibitory effect of cAMP on oxytocin-induced phosphatidylinositide turnover that occurs near the end of pregnancy and may contribute to enhanced myometrial contractile responsiveness near term.

The present study was undertaken in order to identify the mechanism underlying the effect of transforming growth factor-β (TGFβ) on LH receptor (LH-R) expression in rat granulosa cells. Treatment with FSH produced a substantial increase in LH-R mRNA level, and concurrent treatment with increasing concentrations of TGFβ brought about dose-dependent increases in FSH-induced LH receptor mRNA. TGFβ, either alone or in combination with FSH, did not affect intracellular cAMP levels. We then investigated whether the effect of TGFβ and FSH on LH-R mRNA levels results in increased transcription and/or altered mRNA stability. To determine whether the LH receptor 5′-flanking region plays a role in directing LH receptor mRNA expression, the proximal area of the LH receptor 5′-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. FSH (30 ng/ml) significantly enhanced the activity of 1389 base pairs of the LH receptor 5′-flanking region, but treatment with TGFβ did not significantly influence the activity induced by FSH. On the other hand, the decay curves for LH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFβ.

Although it is known that, in the uterus, estrogen receptor α (ERα) is involved in proliferation and progesterone receptor in differentiation, the role of the two other gonadal-hormone receptors expressed in the uterus, androgen receptor (AR) and estrogen receptor β (ERβ), remains undefined. In this study, the involvement of AR in 17β-estradiol (E₂)-induced cellular proliferation in the immature rat uterus was investigated. AR levels were low in the untreated immature uterus, but 24 h after treatment of rats with E₂, there was an increase in the levels of AR and of two androgen-regulated genes, IGF-I and Crisp (cysteine-rich secretory protein). As expected, E₂ induced proliferation of luminal epithelial cells. These actions of E₂ were all blocked by both the antiestrogen tamoxifen and the antiandrogen flutamide. The E₂-induced AR was found by immunohistochemistry to be localized exclusively in the stroma, mainly in the myometrium, where it colocalized with ERα but not with ERβ. ERβ, detected with two different ERβ-specific antibodies, was expressed in both stromal and epithelial cells either alone or together with ERα. Treatment with E₂ caused down-regulation of ERα and ERβ in the epithelium. The data suggest that, in E₂-induced epithelial cell proliferation, ERα induces stromal AR and AR amplifies the ERα signal by induction of IGF-I. Because AR is never expressed in cells with ERβ, it is unlikely that ERβ signaling is involved in this pathway. These results indicate an important role for AR in proliferation of the uterus, where estrogen and androgen do not represent separate pathways but are sequential steps in one pathway.

Calcitonin gene-related peptide (CGRP) is the most potent endogenous vasodilatory peptide, and is involved in the regulation of blood flow to vital organs. We have previously shown that CGRP may be involved in vascular adaptations that occur during pregnancy, and that steroid hormones may be involved in these mechanisms. We hypothesized that endogenous CGRP is required for maintaining blood pressure and fetoplacental growth in pregnant rats, and that progesterone will enhance CGRP effects. The vasodilatory effects of CGRP are known to be inhibited by a competitive CGRP receptor antagonist, the C-terminal fragment CGRP_8-37. In the present study, we investigated whether continuous s.c. infusion of CGRP_8-37 to pregnant rats will reduce fetoplacental growth and increase systolic blood pressure. We also assessed whether progesterone will alter the effects of CGRP_8-37 on blood pressure during postpartum. Groups of five pregnant rats were s.c. infused with varying doses of CGRP_8-37 from Day 17 of pregnancy. Daily systolic blood pressures, pup weight, mortality at term delivery, and fetoplacental weights on Day 20 of gestation were measured. CGRP_8-37 at a dose of 0.083 mg day⁻¹ kg⁻¹ body weight (BW) showed no effects; however, doses of 0.33 and 1.33 mg day⁻¹ kg⁻¹ BW increased (P < 0.05) blood pressure during pregnancy, and these elevated blood pressures persisted during postpartum with the highest dose used. Progesterone (2 mg per injection, twice a day; s.c.) treatment significantly elevated blood pressure in rats infused with CGRP_8-37 during postpartum, suggesting that progesterone regulates CGRP-induced vascular effects. CGRP_8-37 infusion caused significant reductions in pup weight with an increase in mortality rate, and these effects were dose-dependent. Placental and fetal weights were also decreased prior to term on Day 20 of gestation, 72 h after CGRP_8-37 infusion, indicating effects on uteroplacental tissues. Therefore, we suggest that endogenous CGRP plays an important role in maintaining normal fetoplacental development, fetal survival, and vascular adaptations during pregnancy.

A growing body of evidence suggests that the ovary is a site of inflammatory reactions, and thus, ovarian cells could represent sources and targets of the interleukin-1 (IL-1) system. The aim of the present work was to investigate the expression of IL-1α, IL-1β, IL-1ra, IL-1R1, and IL-1R2 genes in equine cumulus cells and oocytes. Moreover, the influence of IL-1β on in vitro maturation of cumulus-oocytes complexes (COCs) was examined. COCs were collected using ultrasound-guided follicular puncture in vivo. Oocytes and cumulus cells were isolated from preovulatory and subordinate follicles and were analyzed either at collection or after in vitro culture. An ultrasensitive reverse transcription-polymerase chain reaction was used to study the expression of IL-1 system members. In order to study the in vitro effect of IL-1β on oocyte nuclear maturation and cumulus expansion, immature COCs collected from subordinate follicles were cultured for 30 h in media containing or not containing IL-1β alone, or in combination with equine luteinizing hormone. Our results indicated that equine oocytes expressed IL-1β and IL-1R2 genes, and that cumulus cells expressed all IL-1 system members except IL-1α. In oocytes, IL-1β expression significantly decreased during in vitro culture compared with immature and mature oocytes analyzed at collection. Similarly, in cumulus cells, in vitro culture decreased IL-1ra and IL-1R1 expression. IL-1β was shown to significantly decrease the gonadotropin-induced nuclear maturation of oocytes. In conclusion, we demonstrated for the first time, in mares, the presence of IL-1β and IL-1R2 transcripts in oocytes and of IL-1β, IL-1ra, IL-1R1, and IL-1R2 in cumulus cells. The regulatory effect of IL-1β on COCs during maturation suggests that the IL-1 system could be of crucial importance for ovarian physiology in the mare.

Somatic cell nuclei of giant pandas can dedifferentiate in enucleated rabbit ooplasm, and the reconstructed eggs can develop to blastocysts. In order to observe whether these interspecies cloned embryos can implant in the uterus of an animal other than the panda, we transferred approximately 2300 panda-rabbit cloned embryos into 100 synchronized rabbit recipients, and none became pregnant. In another approach, we cotransferred both panda-rabbit and cat-rabbit interspecies cloned embryos into the oviducts of 21 cat recipients. Fourteen recipients exhibited estrus within 35 days; five recipients exhibited estrus 43–48 days after embryo transfer; and the other two recipients died of pneumonia, one of which was found to be pregnant with six early fetuses when an autopsy was performed. Microsatellite DNA analysis of these early fetuses confirmed that two were from giant panda-rabbit cloned embryos. The results demonstrated that panda-rabbit cloned embryos can implant in the uterus of a third species, the domestic cat. By using mitochondrial-specific probes of panda and rabbit, we found that mitochondria from both panda somatic cells and rabbit ooplasm coexisted in early blastocysts, but mitochondria from rabbit ooplasm decreased, and those from panda donor cells dominated in early fetuses after implantation. Our results reveal that mitochondria from donor cells may substitute those from recipient oocytes in postimplanted, interspecies cloned embryos.

The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6–9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.

The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at SR (n = 51) and SA (n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using immunoprecipitation and Western ligand blot procedures. IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein, and total IGFBP-2 were not significantly different (P ≥ 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions with higher seminal plasma IGF-I had more total IGFBP-2 per ejaculate (P < 0.01), more morphologically normal sperm (P = 0.05), and higher first-cycle pregnancy rates (P = 0.02). At SA, stallions with higher seminal plasma IGF-I had fewer cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology, and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a ∼20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, β′-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the β′-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.

Proinflammatory cytokines are implicated in the initiation and progression of human labor and delivery, particularly in relation to infection-induced preterm labor. In nongestational tissues, the nuclear factor kappa B (NF-κB) transcription pathway is a key regulator of proinflammatory cytokine release. In these tissues, sulfasalazine (SASP), through its ability to inhibit NF-κB activation, inhibits release of interleukin (IL)-2, IL-12, and tumor necrosis factor (TNF)-α. Therefore, the aim of this study was to investigate whether or not NF-κB activation regulates the formation of proinflammatory cytokines in human gestational tissues. Human placenta, amnion, and choriodecidua (n = 9 separate placentas) were incubated with 10 μg/ml of lipopolysaccharide (LPS) in the absence (control) or presence of SASP (0.1, 1, 5, or 10 mM). After 6 h of incubation, the tissues were collected, and NF-κB DNA binding activity in nuclear extracts was assessed by electromobility shift binding assay. The incubation medium was collected and the release of IL-6, IL-8, and TNF-α was quantified by ELISA. Treatment of placenta, amnion, and choriodecidua with SASP at concentrations 5 mM or greater significantly inhibited the release of IL-6, IL-8, and TNF-α, and NF-κB activation (ANOVA, P < 0.05). The data presented in this study demonstrate that the NF-κB transcription pathway is a key regulator of LPS-stimulated IL-6, IL-8, and TNF-α release from human gestational tissues. The control of NF-κB activation may therefore provide an alternative therapeutic strategy for reducing the release of proinflammatory mediators in infection associated preterm labor.

A novel dodecamer peptide sequence, YLP₁₂, was identified on human sperm that is involved in oocyte binding. We investigated its immunocontraceptive effects in a murine model. A vaccine was prepared by conjugating the synthetic YLP₁₂ peptide with the binding subunit of recombinant cholera toxin. Vaccination of female mice by i.m. or intranasal routes without any additional adjuvant induced a sperm-specific immune response in serum and the vaginal tract that caused a long-term contraceptive state. Fertility was fully regained when antibody reactivity diminished at 305–322 days. The contraceptive effect was also completely reversed voluntarily by intravaginal administration of the peptide. Antibodies affected fertility at the prefertilization stage by inhibiting sperm capacitation and the acrosome reaction, and sperm-oocyte binding. The peptide sequence is an epitope of a 50 ± 5-kDa membrane protein localized on the acrosome and tail of spermatozoa. Thus, the sperm-specific YLP₁₂ is an attractive candidate for contraceptive vaccine.

Extravillous trophoblast (EVT) cells of the human placenta progressively lose their proliferative activity in situ as EVT cell columns migrate into and invade the decidua. It remains unclear whether this is due to a terminal differentiation of EVT cells along the invasive pathway with concomitant loss of proliferative ability, or a negative regulation by decidua-derived factors, or both mechanisms. Our earlier studies provided evidence for a negative regulation by a decidua-derived factor, transforming growth factor (TGF)-β, which inhibited proliferation, migration, and invasiveness of first-trimester EVT cells in vitro. We further discovered that decidua also produces decorin, a proteoglycan that binds TGF-β (and in some cases, inactivates TGF-β), which is colocalized with TGF-β in the decidual extracellular matrix. The present study used in vitro-propagated EVT cell lines to examine whether EVT cells retain their capacity for proliferation after the process of invasion; and whether decorin exerts any effect on EVT cell proliferation, migration, or invasiveness in a TGF-β-dependent or TGF-β-independent manner. We also examined whether trophoblastic cancer (choriocarcinoma) JAR and JEG-3 cells responded to decorin in a similar manner. Proliferation was measured using a colorimetric (MTT) cellularity assay and immunolabeling for the Ki-67 proliferation marker. Migration and invasiveness were measured in transwells by the ability of cells to cross 8-μm pores of polycarbonate membranes in the absence or presence of an additional matrigel barrier. These experiments revealed three points. First, EVT cells retained limited but significant proliferative ability in vitro after invading matrigel. Second, that decorin alone blocked EVT cell proliferation in a dose-dependent manner. This effect remained unaffected in an additional presence of TGF-β, which exerted antiproliferative effects on its own. The antiproliferative effect of decorin was explained by an up-regulation of the p21 protein. Third, that decorin alone or TGF-β alone exerted antimigratory and anti-invasive effects on EVT cells, but the addition of TGF-β to decorin did not alter decorin action. And fourth, that choriocarcinoma cells were resistant to antiproliferative, antimigratory, and anti-invasive effects of decorin. These results suggest 1) that the invasive function of EVT cells is not associated with a terminal differentiation into a noncycling state; 2) that proliferation, migration, and invasiveness of EVT cells within the decidua are independently controlled by two decidual products, TGF-β and decorin (decorin in the decidual extracellular matrix may serve as a storage mechanism for TGF-β in an inactive state and may be activated by EVT cell proteolytic mechanisms, thus preventing overinvasion); and 3) that choriocarcinoma cells are refractory to negative regulation by both decidua-derived factors.