The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5–50 μg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 μg/kg and at 36 h. At the higher dose of sGnRHa (50 μg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1–100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and carbon monoxide, resulting in cGMP production. Recent studies indicate that NO and cGMP influence ovarian functions. However, little information is available regarding the ovarian expression of sGC. The present study examined sGC α₁ and β₁ subunit protein levels in the ovary during postnatal development, gonadotropin-induced follicle growth, ovulation, and luteinization as well as in cultured rat granulosa cells. In postnatal rats, sGC α₁ subunit immunoreactivity was high in granulosa cells of primordial and primary follicles on Day 5 but low in granulosa cells of larger follicles on Days 10 and 19. Theca cells of developing follicles, but not stromal cells, also demonstrated moderate sGC α₁ immunoreactivity. In gonadotropin- treated immature rats, intense sGC α₁ subunit staining was similarly observed in granulosa cells of primordial and primary follicles, but such staining was low in granulosa cells of small antral follicles and undetectable in granulosa cells of large antral and preovulatory follicles. Following ovulation, corpora lutea expressed moderate sGC α₁ immunoreactivity. Similar ovarian localization and expression patterns were seen for sGC β₁, indicating regulated coexpression of sGC subunits. Immunoblot analysis revealed no change in total ovarian sGC α₁ and β₁ subunit protein levels during gonadotropin treatment. Similarly, no effect of FSH on sGC subunit protein levels was apparent in cultured granulosa cells. These findings indicate regulated, cell- specific patterns of sGC expression in the ovary and are consistent with roles for cGMP in modulating ovarian functions.

The dendritic cells and related antigen-presenting cells (APCs) that activate lymphocytes for acquired immunity in the female reproductive tract are not well characterized. The aim of the present study was to examine heterogeneity among uterine APCs in mice and, specifically, to determine whether phenotypically and functionally distinct subpopulations of dendritic cells and macrophages can be identified. Using immunohistochemistry, abundant cells expressing APC-restricted molecules major histocompatibility complex (MHC) class II, F4/80, class A scavenger receptor, macrosialin, and sialoadhesin were evident in estrous mice. Cells expressing the costimulatory molecule B7-2 were rarely observed. Flow cytometric analysis revealed three subpopulations of uterine APCs. Undifferentiated macrophages were F4/80-positive ( ), MHC class II-negative (−) cells, of which 70–80% expressed CD11b, but few expressed class A scavenger receptor, macrosialin, or sialoadhesin. Mature macrophages were F4/80 /MHC class II cells, of which approximately 50% expressed CD11b, class A scavenger receptor, macrosialin, and sialoadhesin. Uterine dendritic cells were F4/ 80−/MHC class II cells, with stimulatory immunoaccessory function relative to uterine macrophages and heterogeneous expression of dendritic markers 33D1, DEC205, CD11c, and CD1. Experiments in ovariectomized mice showed that undifferentiated macrophages were steroid hormone dependent but that mature macrophages and dendritic cells persisted after depletion of ovarian steroid hormones, although with altered phenotypes. In summary, our findings identify three discrete populations of APCs inhabiting the murine uterus and suggest that both mature macrophages and dendritic cells differentiate from undifferentiated macrophage precursor cells. Plasticity in the ontogenetic and functional relationships between uterine dendritic cells and macrophages likely is critical in regulating immune responses conducive to reproductive success.

In the present study, mammary tissues from the fruit bat (Carollia perspicillata) and mouse (Mus musculus) were compared using histological and immunohistochemical methods. Because the female bat exhibits greater reproductive similarities to humans, it might provide a useful animal model for studying mammary physiology and disease with relevance to our own species. In lactating and recently lactating specimens, bat tissue had significantly fewer adipocytes and more collagenous connective tissue compared to the mouse. The proteins Stat5a, keratin 5, Npt2b, and E-cadherin were all similarly localized in mouse and bat mammary tissues taken from lactating animals. The present study demonstrates that whereas the epithelial compartment and the presence of differentiation markers are conserved between the mouse and bat, differences exist in the stromal compartment.

Although gonadotropins have been reported to downregulate FSH-receptor (FSHR) mRNA levels in the ovaries of female rats, the effect of the gonadotropin surge, particularly FSH, on hamster follicular FSHR mRNA levels warrants further examination. The objectives of the present study were to clone and determine the complete FSHR cDNA sequence of the hamster and to delineate the effects of endogenous and exogenous FSH on the steady-state levels of ovarian FSHR mRNA. Complete FSHR cDNA was derived from hamster ovarian total RNA by the strategy of 3′- and 5′-rapid amplification of cDNA ends. Ovaries were obtained before and after the endogenous gonadotropin surge or exogenous FSH administration, and the steady-state levels of FSHR mRNA were assessed by Northern blot hybridization. Cloned FSHR cDNA consists of a reading frame corresponding to exons 1–10 of the human FSHR gene and the 5′- and 3′-untranslated regions. The nucleic acid and amino acid sequences of the reading frame were at least 87% and 92% identical, respectively, to that of human, rat, and mouse FSHR. Furthermore, the amino acid sequence contained seven transmembrane domains characteristic of the FSHR. The steady-state levels of FSHR mRNA increased from estrus (Day 1) to reach a peak on proestrus (Day 4) noon; however, significant attenuation was noted following the gonadotropin surge, which was blocked by phenobarbital. Exogenous FSH also downregulated, both dose- and time-dependently, ovarian FSHR mRNA levels. These data indicate that the nucleic acid sequence of hamster FSHR has been identified and that FSH modulates FSHR mRNA levels in the hamster ovary.

Studies of cloned cattle and mice have resulted in controversies regarding the restoration of eroded telomere length of donor cells by the nuclear transfer process. Little is known about telomere lengths in pigs from either natural reproduction or nuclear transfer. In this study, we measured the telomere lengths in six major porcine organs from animals of different ages, and found that their lengths remained consistent throughout different tissues during fetal stages, and then shortened, in a tissue- specific manner, after birth. Telomeres of skin samples from six cloned transgenic pigs at 4 mo of age did not differ significantly from those of age-matched controls. Two cloned pigs that died shortly after birth had skin telomere lengths equivalent to those of late-stage fetuses.

There is growing evidence that secretin, the first hormone discovered in our history, has functions in the brain other than in the gastrointestinal tract. This article reports for the first time that secretin and its receptor mRNAs are produced in distinct cell types within the epididymis. To test if secretin affects electrolyte transport in the epididymis, we measured short-circuit current (Isc) in cultured epididymal epithelia and found secretin dose-dependently stimulated Isc. Ion substitution experiments and use of pharmacological agents inferred that the stimulated Isc is a result of concurrent electrogenic chloride and bicarbonate secretion. It is further shown that secretin and pituitary adenylate cyclase-activating polypeptide (PACAP) function via totally different mechanisms: 1) PACAP works only from the apical side of the epithelium to stimulate chloride and not bicarbonate secretion, while secretin acts on the apical and basolateral sides to stimulate chloride and bicarbonate secretion. 2) the stimulation by PACAP but not secretin requires local prostaglandin synthesis. By immunocytochemical staining, secretin is localized in the principal cells of the initial segment and caput epididymidis, whereas secretin receptor is present in the principal cells of the proximal as well as the distal part of the epididymis. This pattern of distribution appears to be consistent with the idea that secretin is secreted by the proximal epididymis and acts on the proximal and distal epididymis in an autocrine and paracrine fashion. Its function is to control secretion of electrolytes and water.

Insulin stimulates androgen biosynthesis and the accumulation of CYP17 mRNA and heterogeneous nuclear (hn) RNA in primary cultures of immature swine theca cells. To further assess insulinomimetic transcriptional control, we subcloned 1.007 kilobases (kb) of the 5′-upstream region of the CYP17 gene (−976 to 31 base pairs [bp] to the transcriptional start site) into a firefly-luciferase reporter construct. Insulin drove transcriptional activity of this probe in a time- and dose-dependent fashion, with maximal stimulation of 2.7- to 3.2-fold after insulin exposure (100 ng/ml) for 6 h. Progressive deletional constructs −839, −473, −174, and −75/ 31 bp delineated expected reduction in responsiveness, except paradoxical gain of basal CYP17 promoter activity by the −473/ 31-bp sequence. The latter suggests a possible intervening inhibitory sequence. Elimination of all sequences 5′-upstream to −174 bp markedly reduced basal transcriptional activity and abolished insulin action. Point mutation of a presumptive Sp1-like element located within −193/−180 bp inhibited basal and insulin-stimulated luciferase activity of the full-length promoter fragment by 40% and 67%, respectively. Disruption of a contiguous presumptive AP-2 site produced a comparable outcome. Combined mutation of the Sp1 and AP-2-like elements eliminated basal and insulin-potentiated CYP17 promoter activity. By Western analysis, insulin augmented cognate receptor phosphoprotein concentrations by 31-fold within 10 min. Chemical inhibitors of MEK-activated ERK1/2 attenuated insulin-enhanced CYP17 transcriptional activity by 76–80%. In summary, insulin drives transcriptional activity of a 5′-upstream regulatory sequence (−976 to 31 bp) of the swine CYP17 gene in primary cultures of theca cells, under a minimal requirement for combined activity of proximal (−193/180 bp) Sp1 and AP-2-like elements.

In fertilized mouse eggs, de novo transcription of embryonic genes is first observed during the S phase of the one-cell stage. This transcription, however, is mostly limited to the male pronucleus and possibly uncoupled from translation, making the functional meaning obscure. We found that one-cell mouse embryos respond to the osmotic shock of in vitro isolation with migration of HSF1, the canonical stress activator of mammalian heat shock genes, to pronuclei and by transient transcription of the hsp70.1, but not hsp70.3 and hsp90, heat shock genes. Isolated growing dictyate oocytes also display a nuclear HSF1 localization, but, in contrast with embryos, they transcribe both hsp70.1 and hsp70.3 genes only after heat shock. Intranuclear injection of double-stranded oligodeoxyribonucleotides containing HSE, GAGA box or GC box consensus sequences, and antibodies raised to transcription factors HSF1, HSF2, Drosophila melanogaster GAGA factor, or Sp1 demonstrated that hsp70.1 transcription depends on HSF1 in both oocytes and embryos and that Sp1 is dispensable in oocytes and inhibitory in the embryos. Hsp70.1 thus represents the first endogenous gene so far identified to be physiologically activated and tightly regulated after fertilization in mammals.

One of the mysteries of pregnancy is why a mother does not reject her fetuses. Cytokine-modulation of maternal-fetal interactions is likely to be important. However, mice deficient in transforming growth factor-β1 (TGFβ1) and other cytokines are able to breed, bringing this hypothesis into question. The phenotype of TGFβ1 null-mutant mice varies with genetic background. We report here that, in outbred mice, the loss of TGFβ1- deficient embryos is influenced by the parity of their mother. This is consistent with the loss of mutants being due to immune rejection. An inbred line of TGFβ1 ^/− mice that supported TGFβ1-deficient fetuses had high levels of TGFβ1 in their plasma. Analysis of the amniotic fluids in this line indicated that biologically relevant levels of maternal TGFβ1 were present in the TGFβ1^−/− fetuses. These data are consistent with maternal and fetal TGFβ1 interacting to maintain pregnancy, within immune-competent mothers.

We previously identified Gasz (a germ cell-specific gene encoding a protein containing four ankyrin repeats, a sterile-α motif, and a basic leucine zipper) in six mammalian species. Here, we report GASZ orthologs in pufferfish (Fugu rubripes), zebrafish (Danio verio), and frog (Xenopus laevis). Sequences of the three Gasz cDNAs were determined by database mining and 5′- and 3′-rapid amplification of cDNA ends (RACE) followed by sequencing. The three orthologous vertebrate genes encode proteins structurally similar to mammalian GASZ and contain the characteristic four ankyrin repeats (ANKs) and sterile-α motif (SAM). Their ANK and SAM domains share 55– 74% and 38–55% amino acid identity with those in human GASZ, respectively. Similar to human and mouse Gasz genes, pufferfish Gasz is composed of 13 exons, spanning approximately 12 kilobases, and flanked by Cftr at its 5′-end and Wnt2 at its 3′-end. Northern and Western blot analyses detect frog Gasz expression only in testis and ovary. In situ hybridization and immunohistochemical analyses show that frog Gasz mRNA and protein expression is confined to pachytene spermatocytes in the testis and to oocytes in the ovary. In frog oocytes, GASZ protein appears to localize to a cytoplasmic structure resembling the Balbiani body, a postulated mRNA transport organizer in the cytoplasm. The high evolutionary conservation and germ cell specificity suggest that GASZ plays an essential role in gametogenesis. The data presented here are important for future studies of the physiological roles of GASZ using fish and amphibians as animal models.

Hyperactivated sperm motility is characterized by high-amplitude and asymmetrical flagellar beating that assists sperm in penetrating the oocyte zona pellucida. Other functional changes in sperm, such as activation of motility and capacitation, involve cross talk between the cAMP/PKA and tyrosine kinase/phosphatase signaling pathways. Our objective was to determine the role of the cAMP/protein kinase A (PKA) signaling pathway in hyperactivation. Western blot analyses of detergent extracts of whole sperm and flagella were performed using antiphosphotyrosine antibody. Bull sperm capacitated by 10 μg/ml heparin and/or 1 mM dibutyryl-cAMP plus 100 μM 3-isobutyl-1-methylxanthine exhibited increased protein tyrosine phosphorylation without becoming hyperactivated. Procaine (5 mM) or caffeine (10 mM) immediately induced hyperactivation in nearly 100% of motile sperm but did not increase protein tyrosine phosphorylation. After 4 h of incubation with caffeine, sperm expressed capacitation-associated protein tyrosine phosphorylation but hyperactivation was significantly reduced. Sperm initially hyperactivated by procaine or caffeine remained hyperactivated for at least 4 h in the presence of Rp-cAMPS (cAMP antagonist) or PKA inhibitors H-89 or H-8. Pretreatment with inhibitors also failed to block induction of hyperactivation; however, the inhibitors did block protein tyrosine phosphorylation when sperm were incubated with capacitating agents, thereby verifying inhibition of the cAMP/PKA pathway. While induction of hyperactivation did not depend on cAMP/PKA, it did require extracellular Ca² . These findings indicate that hyperactivation is mediated by a Ca² signaling pathway that is separate or divergent from the pathway associated with acquisition of acrosomal responsiveness and does not involve protein tyrosine phosphorylation downstream of the actions of procaine or caffeine.

The developmental potential of oocytes from prepubertal cattle is decreased, compared with those from their adult counterparts. The aim of the present study was to improve the developmental capacity of oocytes from prepubertal cattle by either systemic application of recombinant bovine somatotropin (rbST) or intraovarian injection of insulin-like growth factor-I (IGF-I). Blastocyst yields and the mRNA expression pattern (relative abundance, RA) of three putative marker genes (i.e., glucose transporter-1, Glut-1; eukaryotic translation initiation factor-1A, eIF1A, and upstream binding factor, UBF) were selected as criteria to determine the success of the treatments. At 6–7 mo of age, 30 healthy Holstein calves were randomly assigned to three experimental groups. The first group served as control and received an intraovarian injection of 0.6 ml acetic acid. The second group received a single s.c. injection of 500 mg of rbST. The third group received an intraovarian injection of 6 μg recombinant human IGF-I. During the following 2 wk, follicles were aspirated four times via transvaginal ultrasound-guided technology. All animals were i.m. injected with 60 mg FSH 48 h prior to each aspiration. The treatments were repeated with the same animals at 9–10, 11–12, and 14–15 mo of age. For comparison, five adult cows were each i.m. injected with 100 mg FSH and underwent oocyte retrieval. The proportion of oocytes considered to be developmentally competent was higher in cows than calves (65% vs. 58%, 50%, 52%) for the control, rbST, and IGF-I groups, respectively. The rate of blastocysts was similar in IGF-I-treated calves and cows (28% and 25%) and was higher (P ≤ 0.05) than in the controls and the rbST group (11% and 16%). The RA for Glut-1 was lower (P ≤ 0.05) in two- to four- cell embryos from calves, compared with cows. At the 8- to 16- cell stage, Glut-1 RA was similar in IGF-I-treated calves and cows. The RA for eIF1A was higher (P ≤ 0.05) in 8- to 16-cell embryos derived from cows than those from the control group. Results show that IGF-I intraovarian injection increased blastocyst yields and mRNA expression of Glut-1 and eIF1A to levels found in embryos produced from adult cows. This treatment may at least partially overcome the developmental deficiency of oocytes derived from calves and could be a step forward toward the use of prepubertal animals in breeding programs aimed at shortening the generation interval.

Parthenogenesis (PA) of the oocyte is essential to a number of oocyte- or embryo-related technologies such as intracytoplasmic sperm injection and cloning by nuclear transfer. This study investigated the onset and frequency of apoptosis in PA- porcine embryos and the morphological changes that conform to the general criteria of apoptotic cell death by using a terminal deoxynucleatidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. PA embryos had a higher degree of apoptotic cell death during in vitro culture, a lower cleavage rate (45% vs. 71%), and a lower development rate to the blastocyst stage (16% vs. 29%), relative to in vitro fertilization (IVF). The earliest positive TUNEL signal in the PA embryos was detected on Day 6, 1 day later than that in IVF embryos. Apoptosis in PA embryos increased from 15% of the embryos on Day 6 to 29% on Day 8. The mean level of apoptosis of the PA embryos was statistically higher than that of IVF embryos, except on Day 5. In particular, apoptosis in PA embryos was twice that of IVF embryos on Day 6 (15% vs. 6.7%) and Day 8 (29% vs. 13%). The mean cell number in PA blastocysts was significantly lower than that of IVF blastocysts, whereas the percentage of apoptosis in PA blastocysts was significantly higher than that of IVF blastocysts. There was a high percentage of haploid (62.5%) PA blastocysts. The ploidy may contribute to a high level of apoptosis. These results may help to explain the mechanism of parthenogenetic developmental failure and may lead to methods that will improve parthenogenetic development.

We investigated whether leptin can suppress the prepartum activation of the fetal hypothalamus-pituitary-adrenal (HPA) axis and delay the timing of parturition in the sheep. First, we investigated the effects of a 4-day intravascular infusion of recombinant ovine leptin (n = 7) or saline (n = 6) on fetal plasma adrenocorticotropic hormone (ACTH) and cortisol concentrations, starting from 136 days gestation (i.e., at the onset of the prepartum activation of the fetal HPA axis. The effects of a continuous intrafetal infusion of leptin (n = 7) or saline (n = 5) from 144 days gestation on fetal plasma ACTH and cortisol concentrations and the timing of delivery were also determined in a separate study. There was an increase in fetal plasma ACTH (P < 0.01) and cortisol (P < 0.001) concentrations when saline was infused between 136–137 and 140–141 days gestation. Plasma ACTH and cortisol concentrations did not rise, however, when leptin was infused during this period of gestation. When leptin was infused after 144 days gestation, there was no effect of a 4- to 5-fold increase in circulating leptin on fetal ACTH concentrations. In contrast, leptin infusion from 144 days gestation suppressed (P < 0.05) fetal plasma cortisol concentrations by around 40% between 90 and 42 h before delivery. There was no difference, however, in the length of gestation between the saline- and leptin-infused groups (saline infused, 150.2 ± 0.5 days; leptin infused, 149.8 ± 1.0 days). In saline-infused fetuses, there was a significant negative relationship between the plasma concentrations of cortisol (y) and leptin (x) between 138 and 146 days gestation (y = 81.4 − 7.7x, r = 0.38, P < 0.005). This study provides evidence for an endocrine negative feedback loop between leptin and the HPA axis in fetal life.

The vascular relaxation sensitivity to calcitonin gene-related peptide (CGRP) is enhanced during pregnancy, compared with nonpregnant human and rat uterine arteries. In the rat uterine artery, two types of CGRP receptors have been shown to coexist, CGRP-A receptor, which is a complex of calcitonin receptor-like receptor (CRLR), and receptor activity–modifying protein (RAMP₁) and CGRP-B receptor, which is different from CRLR. In the present study, we hypothesized that: 1) CGRP-induced vasorelaxation in rat uterine artery is mediated through CGRP-A receptor and 2) N-terminal (Nt) domain of CRLR (Nt-CRLR) has a major contribution in ligand binding and mediating CGRP- induced relaxation effects in rat uterine artery. Polyclonal antibodies against Nt-domain of CRLR and RAMP₁ (Nt-RAMP₁) were raised in rabbits and characterized for their specificity and were used to inhibit CGRP-induced vasorelaxation in rat uterine artery. For vascular relaxation studies, uterine arteries from Day 18 pregnant rats were isolated, and responsiveness of the vessels to CGRP was examined with a small vessel myograph. CGRP (10⁻¹⁰ to 10⁻⁷ M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in Day 18 pregnant rat uterine arteries. These effects were significantly (P < 0.05) inhibited when uterine arteries were incubated with the antibody raised against Nt-CRLR (PD₂ = 6.75 ± 0.20) and were totally abolished in presence of antibodies for both Nt-CRLR and Nt-RAMP₁ (PD₂ = 6.14 ± 0.35). In contrast, a monoclonal antibody for CGRP-B receptor had no effect on CGRP-induced rat uterine artery relaxation. These studies suggest that CGRP effects in rat uterine artery are mediated through CGRP-A receptor and that Nt-domain of CRLR may play a predominant role in CGRP binding and thus in causing CGRP-induced uterine artery relaxation.

Insulin-like growth factor-I (IGF-I) is involved in the regulation of ovarian follicular development and has been shown to potentiate the FSH responsiveness of granulosa cells from preantral follicles. The aim of the present study was to investigate the effect of IGF-I during preantral follicular culture on steroidogenesis, subsequent oocyte maturation, fertilization, and embryo development in mice. Preantral follicles were isolated mechanically and cultured for 12 days in a simplified culture medium supplemented with 1% fetal calf serum, recombinant human FSH, transferrin, and selenium. In these conditions, follicles were able to grow and produce oocytes that could be matured and fertilized. The first experiment analyzed the effect of different concentrations of IGF-I (0, 10, 50, or 100 ng/ml) added to the culture medium on the follicular survival, steroidogenesis, and the oocyte maturation process. The presence of IGF-I during follicular growth increased the secretion of estradiol but had no effect on the subsequent oocyte survival and maturation rates. In the second experiment, IGF-I (0 or 50 ng/ml) was added to the culture medium during follicular growth, oocyte maturation, or both, and subsequent oocyte fertilization and embryo development rates were evaluated. Oocyte fertilization rates were comparable in the presence or absence of IGF-I. However, the blastocyst development rate was enhanced after follicular culture in the presence of IGF-I. Moreover, the total cell number of the blastocysts observed after differential labeling staining was also higher when follicles were cultured or matured in the presence of IGF-I.

In penaeid shrimp, cortical rods (CRs) are formed in peripheral crypts of the oocyte after completion of yolk accumulation; subsequently the CRs are utilized as a source of jelly materials that surround fertilized eggs. In our previous study, of five major components, three CR proteins displayed quite similar immunological characteristics. In this study, cDNA sequences and developmental expression profiles at both transcriptional and protein levels were examined to elucidate the molecular characteristics of CR proteins and the process of CR formation. Sequencing cDNAs exhibited the presence of three related forms that have identical sequences except for the loss of 246 and 369 bp in medium and short forms, respectively, suggesting that a single gene generates three transcriptional variants corresponding to the three CR proteins. Their deduced amino acid sequences revealed similarities to those of extracellular matrix proteins in a thrombospondin (TSP) 3,4/cartilage oligomeric protein family, and thereby the CR proteins were designated mjTSP. Semiquantitative analysis by real-time polymerase chain reaction revealed the presence of mjTSP transcripts, at similar levels, in immature, vitellogenic, and mature ovaries. Furthermore, in situ hybridization localized the majority of transcripts in previtellogenic oocytes in ovaries at all developmental stages. By the Western blot, on the other hand, mjTSP proteins were undetectable in immature ovaries but became obvious at the early vitellogenic stage. The immunosignals were enhanced during vitellogenic stages and maintained a high intensity in mature ovaries. Thus, transcription, translation of mjTSP, and formation of the CR structure occurred at different stages of ovarian development.

The most common parameters used to evaluate sperm quality are motility rate and duration and fertilization ability. In this study, chemical and biochemical parameters of sea bass (Dicentrarchus labrax) sperm were investigated to find an alternative method for evaluating sperm fertilization ability before and after cryopreservation. The biochemical and chemical analyses were performed with fresh and frozen-thawed sperm and seminal plasma. To cryopreserve sperm, 250-μl straws were used. Fertilization ability was evaluated by inseminating eggs (obtained from hormonally stimulated females) with fresh and cryopreserved sperm. The results revealed a linear relationship (P < 0.05) between semen fertilization capacity and some seminal plasma (β-d-glucuronidase activity, potassium concentration) and sperm (ATP concentration, aspartate aminotransferase activity) parameters. Variations in semen fertilization rate could be best described by two multiple regression models: one including the sperm parameters and another including the seminal plasma parameters. For practical application, the use of simple regression models is of value. Fertilization rate in both fresh and cryopreserved sperm was reliably predicted by determining the ATP concentration or the β-d-glucuronidase activity or both.

The cholesterol-depleting drug methyl-β-cyclodextrin (Me-β- CD) was tested for its effects on amphibian oocyte maturation, cholesterol depletion, and low-density membrane recovery. Progesterone-induced oocyte maturation was accelerated by pretreatment of cells with 5–50 mM Me-β-CD in a dose-dependent manner. Treatment of oocytes with 50 mM Me-β-CD alone was sufficient to induce germinal vesicle breakdown, stimulate formation of meiotic spindles, and stimulate phosphorylation of mitogen-activated protein kinase over time courses longer than those observed after progesterone treatment. After short-term (30 min) labeling of oocytes with [³H]cholesterol, 30–90 min of treatment with 5–50 mM Me-β-CD removed 50%–70% of cell- associated label, and cholesterol depletion was not observed with α-cyclodextrin. After long-term (20–23 h) labeling of oocytes with [³H]cholesterol, Me-β-CD treatment resulted in dose- dependent cholesterol depletion in the 5–50 mM range, and 50 mM Me-β-CD removed approximately 50% of cell-associated label after 9 h. Treatment of oocytes with 5–50 mM Me-β-CD also decreased recovery of low-density membrane by detergent- free sucrose gradient centrifugation. These results implicate cholesterol and low-density membrane domains in the signaling mechanisms leading to germinal vesicle breakdown in amphibian oocytes.

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT (1,1-dichlorodiphenyltrichloroethane), is a persistent hormonally active environmental toxicant that has been found in human serum and follicular fluid. The objective of this study was to determine whether DDE can alter free calcium ion concentrations in the cytosol ([Ca² ]_cyt) of human granulosa cells. Changes in [Ca² ]_cyt in single cells loaded with Fura-2 were studied using a dynamic digital Ca² imaging system. At a concentration of 100 ng/ml, DDE stimulated small elevations of [Ca² ]_cyt accompanied by Ca² oscillations. At 1 μg DDE/ml, there was a biphasic Ca² response with marked elevations of [Ca² ]_cyt over time. In Ca² -free medium, cells showed an initial small elevation of [Ca² ]_cyt, which was magnified after addition of Ca² to the medium. Washing the cells after DDE treatment failed to remove the elevated [Ca² ]_cyt and oscillations, both of which were eliminated by addition of EGTA. ATP also induced [Ca² ]_cyt elevations and oscillations, and these effects were potentiated when DDE was added. FSH induced transient [Ca² ]_cyt elevations, whereas hCG caused a prolonged elevation and marked oscillations in [Ca² ]_cyt. These results suggest that DDE at concentrations normally found in human tissues induces elevations in [Ca² ]_cyt in granulosa-lutein cells. Our data therefore highlight a novel mechanism through which DDE can alter endocrine homeostasis and possibly act as an endocrine toxicant.

Maternal-zygotic transition (MZT) is a complex phenomenon characterized by the initiation of transcription in the embryo and the replacement of maternal mRNA with embryonic mRNA. In order for this to occur, transcriptional activation requires various factors and conditions. Our hypothesis is that the lack of transcription in the bovine pre-MZT embryo is due, in part, to an incomplete or dormant transcriptional apparatus. Therefore, in accordance with this hypothesis, functioning transcriptional mechanisms should appear in the eight-cell bovine embryo to facilitate embryonic transcription during the MZT. With this in mind, we examined the presence of selected transcription factors during preimplantation embryo development to establish how their transcript levels change in bovine pre-MZT embryos. To achieve this goal, real-time reverse transcription-polymerase chain reaction was used to quantify the mRNA level of several different transcription factors (YY1, HMGA1, RY-1, P300, CREB, YAP65, HMGN1, HMGB1, NFAR, OCT-4, TEAD2, ATF-1, HMGN2, MSY2, and TBP) in germinal vesicle (GV) and metaphase II (MII) bovine oocytes and in two-, four-, eight-cell, and blastocyst stage embryos produced in vitro. Our results demonstrate that all genes examined can be grouped into five different categories according to their mRNA expression patterns at the developmental stages observed. To summarize, all transcription factors studied were present in pre-MZT embryos and the expression pattern of many of them suggest a potential role in MZT.

Previous data showed that glycodelin-A from amniotic fluid and glycodelin-F from follicular fluid inhibited sperm-zona pellucida binding. Solubilized zona pellucida reduced the binding of glycodelin-F to sperm extract dose dependently. This study demonstrated that the zona pellucida proteins also reduced the binding of glycodelin-A to sperm extract. Ionophore-induced acrosome reaction reduced the binding of iodinated glycodelin-A and -F to sperm, indicating that the glycodelin-binding sites are on the outer acrosomal membrane or on the sperm plasma membrane overlying the acrosome. While the binding of glycodelin-A to sperm was suppressed by mannose and fucose neoglycoproteins, that of glycodelin-F was also reduced by acetylglucosamine neoglycoprotein. Pretreatment of sperm with inhibitors of mannosidase and acetylglucosaminidase reduced the binding of glycodelin-F to sperm. On the other hand, inhibitor of mannosidase but not of acetylglucosaminidase inhibited the binding of glycodelin-A. In a competition binding assay, mannosidase reduced both glycodelin-A and -F binding whereas acetylglucosaminidase reduced only glycodelin-F binding. While fucosidase reduced the binding of both glycodelins, fucosidase inhibitor was marginally active in suppressing the binding of glycodelins to human sperm. Among the selectins tested, only E-selectin had a slight inhibitory effect on the binding of glycodelin-A to sperm. The binding of glycodelin-F was unaffected by selectins and their antibodies. In conclusion, the binding of glycodelin-A to sperm involves mannose, fucose, and possibly E- selectin residues, while that of glycodelin-F involves mannose, fucose, and N-acetylglucosamine but not the selectin residue.

A full-length (4021 base pair [bp]) cDNA encoding a polypeptide (844 amino acids) with a predicted mass of 93 kDa and other characteristic structural features of a vertebrate vitellogenin receptor (VgR) was isolated from a white perch (Morone americana) ovarian cDNA library. Northern blotting performed using a specific digoxygenin-labeled VgR cDNA probe revealed a distinct ∼4.1 kilobase (kb) hybridization signal in an mRNA preparation obtained from previtellogenic perch ovaries. The deduced amino acid sequence of the perch VgR was 89% and 82% identical, respectively, to that of the tilapia and rainbow trout. Because it possessed an eight-repeat ligand-binding domain (LR8) but lacked an O-linked sugar domain (−), the perch VgR was identified as a non-O-linked form of VgR (LR8−). Unlike the case in other vertebrates investigated, including tilapia and trout, no species of mRNA encoding an O-linked form of VgR (LR8 ) could be detected when perch ovarian or liver mRNA reverse transcripts or cDNA libraries were screened by PCR using primer sets flanking the putative O-linked sugar domain. These novel findings call into question the assumptions that an LR8 splice variant of the VgR always is dominantly present in somatic tissues and exists at lower levels in ovarian tissues to sequester lipoproteins distinct from Vg. A SYBR-green-based real-time reverse transcription-polymerase chain reaction assay was developed and used to quantitatively measure VgR expression in gonadal and somatic tissues, for the first time in any vertebrate. The main site of perch VgR mRNA expression was the ovary and the highest level of VgR mRNA expression was in ovaries whose largest follicles contained previtellogenic oocytes. Expression of VgR mRNA decreased with oocyte growth during vitellogenesis and was very limited in ovulated eggs. These quantitative results verify the concept that growing oocytes must extensively recycle LR8− forms of the VgR.

Spermatogenesis originates in spermatogonial stem cells, which have the unique mode of replication. It is considered that a single stem cell can produce two stem cells (self-renewing division), one stem and one differentiating (asymmetric division), or two differentiating cells (differentiating division). However, little is known regarding how each type of division is regulated. In this investigation, we focused on the analysis of self- renewing division and examined the effect of the pituitary gland using two models of stem cell self-renewing division. In the first experiment using newborn mice, the administration of GnRH- analogue, which represses the release of gonadotropin, reduced the number of stem cells during postnatal testicular development, suggesting that the pituitary gland enhances stem cell self- renewing division. In the second experiment, however, the number of stem cells increased dramatically in hypophysectomized adult recipients after spermatogonial transplantation. Thus, the pituitary gland affects the self-renewing division of stem cells, but these contradictory results suggest that its role may be different depending on the stage of the testicular development.

The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300–500/cm²) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-α (TGFα), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGFα, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except α₆-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.

In rodents, changes in gene expression during spermatogenesis can be monitored by sampling testis from each day during postnatal development. However, changes in gene expression at the tissue level can reflect changes in the concentration of an mRNA in a specific cell type, changes in volume of specific cells, or changes in the cell-type composition. This reflects the cellularity of the tissue. Here we have combined techniques that assess the expression profiles of genes at the whole-tissue level, differential display and DNA array, and, at the level of cellularity, in situ hybridization. Combining results from these techniques allows determination of the cell-type-specific gene-expression patterns of many genes during spermatogenesis. Differential display was used to determine expression profiles with high sensitivity and independent of prior knowledge of the sequence, whereas DNA arrays quickly assess the expression profiles of all the genes. This identified three groups of gene-expression profiles. The major group corresponds to genes that are upregulated in spermatocytes during either the mid- or late- pachytene phase of spermatogenesis (stages VII–XI). This pachytene cluster was gradually extinguished in the later spermatid stages but was followed by another cluster of genes expressed in spermatids. Finally, a group of genes was downregulated during spermatogenesis and probably expressed in nongerm cells. We believe that expression of most genes can be described by a combination of these cell-type-specific expression patterns.

1-β-d-Arabinofuranosylcytosine (Ara-C), a DNA-damaging agent, severely inhibits fetal growth and has teratogenicity. Recently, we reported that Ara-C also causes placental growth retardation and increases placental apoptosis. The aim of the present study is to elucidate the mechanisms of placental injury induced by genotoxic stress and involvement of p53, which mediates apoptosis and cell-cycle arrest after DNA damage. We injected Ara-C into pregnant rats on Day 13 of gestation and examined the placentas from 1 to 48 h after the administration. Terminal deoxynucleotidyltransferase-mediated dUTP end-labeling (TUNEL) revealed that the apoptosis of trophoblastic cells in the placental labyrinth zone increased from 3 h after the treatment and peaked at 6 h before returning to control levels at 48 h. An increase in cleaved caspase-3 immunoreactivity was also detected at 6 h. Proliferative activity as measured by immunohistochemistry for topoisomerase IIα and by mitotic index significantly decreased after the treatment in the labyrinth zone. Immunoreactivity for p53 protein in the placental labyrinth zone was remarkably enhanced and peaked at 3 h after treatment, although no increase in p53 mRNA expression was detected with a reverse transcription- polymerase chain reaction. Regarding p53 target genes, p21, cyclinG1, and fas mRNA levels increased significantly and peaked at around 9 h after the treatment. These results indicate that Ara-C would induce apoptosis and impair cell proliferation in the placental labyrinth zone, and p53 and its transcriptional target genes may play an important role in the pathogenesis of the Ara-C-induced placental toxicity.

Recent evidence suggests that the ovine premammillary hypothalamic area (PMH) is an important target for the pineal hormone, melatonin, and its role in seasonal reproduction. In rodents, the PMH is a complex region consisting of several cell groups with differing neurochemical content and anatomical connections. Therefore, to obtain a better understanding of the potential neural targets for melatonin in this area of the sheep brain, we have undertaken a detailed anatomical characterization of the PMH, including its nuclear divisions and the location of neuropeptide/neurotransmitter cells within them. By combining immunocytochemistry for NeuN, a neuronal marker, with Nissl staining in anestrous, ovariectomized, estradiol-treated ewes, we identified three nuclei within the PMH: a caudal continuation of the hypothalamic arcuate nucleus (cARC), the ventral division of the premammillary nucleus (PMv), and the ventral tuberomammillary nucleus (TMv). The cARC contained neurons that were immunoreactive for tyrosine hydroxylase, dynorphin, estrogen receptor α, cocaine- and amphetamine-regulated transcript peptide (CART), and nitric oxide synthase (NOS). The PMv was also characterized by the presence of cells that contained NOS and CART, although the size of these cells was larger than that of their corresponding phenotype in the cARC. By contrast, in the TMv, of the markers examined in the present study, only fibers immunoreactive for orexin were seen. Thus, the ovine PMH is a heterogeneous region comprised of three subdivisions, each with distinct morphological and neurochemical characteristics. This anatomical map of the PMH provides a basis for future studies to determine the functional contribution of each component to the influence of melatonin on seasonal reproduction.

To date, the laboratory mouse is the only mammal in which freeze-dried spermatozoa have been shown to support full-term development after microinjection into oocytes. Because spermatozoa in mice, unlike in most other mammals, do not contribute centrosomes to zygotes, it is still unknown whether freeze-dried spermatozoa in other mammals are fertile. Rabbit sperm was selected as a model because of its similarity to human sperm (considering the centrosome inheritance pattern). Freeze- drying induces rabbit spermatozoa to undergo dramatic changes, such as immobilization, membrane breaking, and tail fragmentation. Even when considered to be “dead” in the conventional sense, rabbit spermatozoa freeze-dried and stored at ambient temperature for more than 2 yr still have capability comparable to that of fresh spermatozoa to support preimplantation development after injection into oocytes followed by activation. A rabbit kit derived from a freeze-dried spermatozoon was born after transferring 230 sperm-injected oocytes into eight recipients. The results suggest that freeze-drying could be applied to preserve the spermatozoa from most other species, including human. The present study also raises the question of whether rabbit sperm centrosomes survive freeze-drying or are not essential for embryonic development.

Mos is a MAPK kinase kinase with an expression that is highly restricted to the gonads. Its function is mainly associated to the meiotic metaphase II arrest occurring during female gametogenesis, whereas to our knowledge, its role during spermatogenesis has not yet clarified. In the present paper, we report the isolation of c-mos cDNA and the identification of a 60-kDa Mos protein from the testis of the anuran amphibian, Rana esculenta. Both the transcript and the protein are always present at low levels in the testis during the frog annual sexual cycle, with single significant peaks of expression in March and May, respectively. Mos is mainly localized in the cytoplasm of primary and secondary spermatogonia (SPG). Therefore, we have used treatments with ethane-dimethane sulphonate (EDS), which blocks spermatogonial mitosis in frogs. Four days after a single EDS injection, Mos expression in SPG highly increases concomitantly with the temporary arrest of mitosis. From 8 to 28 days after the injection, the normal proliferative activity of SPG is restored, and Mos expression gradually decreases to control levels. These results strongly indicate that the c-mos proto-oncogene exerts a new role associated to the regulation of spermatogonial proliferation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely toxic, persistent environmental contaminant that disrupts normal development in laboratory animals. In our earlier study, we found that exposure of preimplantation embryos to TCDD markedly induced cytochrome P4501A1 mRNA at the blastocyst stage. In the present study, to determine whether exposure of preimplantation embryos to TCDD affects fetal growth, we exposed preimplantation embryos to TCDD from the 1-cell stage to the blastocyst stage and then transferred them to unexposed recipient mice. On Embryonic Day 14, the fetuses exposed to TCDD during the preimplantation stage weighed less than the fetuses in the unexposed control group. Real-time reverse transcription-polymerase chain reaction analysis revealed that exposure of preimplantation embryos to TCDD tended to decrease the expression levels of the imprinted genes H19 and Igf2 (insulin-like growth factor 2 gene). Use of bisulfite genomic sequencing demonstrated that the methylation level of the 430- base pair H19/Igf2 imprint control region was higher in TCDD- exposed embryos and fetuses than in the controls, and methyltransferase activity was also higher in the TCDD-exposed embryos than in the controls. To our knowledge, the present study is the first to provide evidence that TCDD exposure at the preimplantation stage alters the genomic DNA methylation status of imprinted genes, influences the expression level of imprinted genes, and affects fetal development.

Forty-week-old male broiler breeders were used in two experiments. Males were reared as recommended by the breeder, housed in individual cages, and cannulated to facilitate blood sampling. In experiment 1, blood samples were collected at 10- min intervals for 4 h commencing the day of cannulation (Day 0) and for 12 h on each of Days 1 and 2. In experiment 2, blood samples were collected at 10-min intervals for 8 h on Day 1. After centrifugation, plasma was stored at −20°C until LH, FSH (experiment 1 and 2), testosterone, and corticosterone (experiment 1) concentrations were determined by RIA. Different statistical methods used to identify hormone secretion profiles revealed a characteristic pulsatile pattern of LH and FSH in plasma. However, LH pulses were more frequent and had greater amplitude than FSH pulses. Less than 32% of the FSH pulses were associated with LH episodes. Conversely, the association between LH and testosterone pulses averaged 83% in birds with testis weight greater than 10 g. Concentrations of corticosterone tended to increase after cannulation and remained elevated for only 3–4 h. Our data indicate that LH, FSH, and testosterone secretion is pulsatile in male broiler breeders. Additionally, LH pulses are associated with testosterone episodes but not with FSH pulses. The pulsatile pattern of FSH secretion, which is unique from those of LH, in adult males suggests that FSH secretion is independently regulated in the adult male fowl.

The mouse is an excellent model for studying the genetic basis of placental development, but analyses are restricted by the lack of quantitative data describing normal murine placental structure. This study establishes a technique for generating such data, applies stereological techniques on systematic uniform random sections of placentas between E12.5—E18.5 of gestation (E1.0 = day of the vaginal plug), and considers the results in the context of development of the labyrinth zone. Half of each placenta was wax embedded and exhaustively sectioned to determine absolute volumes of the labyrinth zone (Lz), junctional zone (Jz), and decidua using the Cavalieri principle. The other half was resin embedded and 1-μm sections were used to generate all volume, surface, and length densities within the Lz. Maximum placental volume is reached by E16.5, whereas the Lz volume fraction increases until E18.5 at the expense of the Jz and decidua. Within the Lz, the absolute volume and surface area of maternal blood spaces (MBS) expand rapidly between E14.5 and E16.5, with no increase thereafter. In contrast, fetal capillary development is linear and continues for longer than that of the MBS. The interhemal membrane separating maternal and fetal circulations undergoes thinning prior to expansion of maternal and fetal surface areas, achieving a harmonic mean thickness of 4.39 μm by E18.5. The specific diffusion capacity for oxygen of the interhemal membrane is maximal by E16.5, which may be necessary to support rapid fetal growth until the end of gestation.

Retinoic acid (RA) was recently shown to modify testosterone secretion of the fetal testis in vitro. We characterized this effect by culturing rat testes explanted at various ages, from Fetal Day 14.5 to Postnatal Day 3. In basal medium, RA inhibited, in a dose-dependent manner, both basal and acute LH-stimulated testosterone secretion by testes explanted on Fetal Days 14.5, 15.5, and 16.5. It had no effect on testes from older animals. The negative effect of RA did not result from a diminution in the number of Leydig cells but from a decrease in P450c17 mRNA levels and in LH-stimulated cAMP production. However, the RA-induced decrease in P450C17 mRNA levels was also observed with neonatal testes, suggesting that this enzymatic step is no longer rate limiting at this developmental stage. To study the physiological relevance of RA effects, we used fetuses and neonates issued from mothers fed a vitamin A-deficient (VAD) diet, resulting in a threefold decrease of plasma retinol concentration. On Fetal Day 18.5 and on Posnatal Day 3, testosterone secretion by the testis ex vivo was significantly increased in VAD animals. This shows that the endogenous retinol inhibits differentiation and/or function of fetal Leydig cells before Fetal Day 18.5 and is required for the normal regression of fetal Leydig cell function that occurs after Fetal Day 18.5. In conclusion, our results show that retinoids play a negative role on the steroidogenic activity during the differentiation of rat fetal Leydig cells.

During early pregnancy, an environment of relative low oxygen tension is essential for normal embryonic and placental vasculature. In low-oxygen conditions, the hypoxic-inducible factor-1 (HIF-1), composed of α and β subunits, controls the expression of a number of genes such as vascular endothelial growth factor (VEGF), a key angiogenic factor. The recent studies in some tumor cells have found that the labile component, HIF-1α, is not only activated by hypoxia but also by peptides such as interleukin-1 (IL-1) in normoxia. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to IL-1β stimulated the expression of HIF-1α protein. Meanwhile, IL-1β also induced the secretion of VEGF in normal human cytotrophoblast cells. Our data indicated that IL-1β induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Moreover, treatment of cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited the stimulation of HIF-1α protein expression and VEGF secretion by IL-1β. These data indicate that, in normal human cytotrophoblast cells, IL-1β induces HIF- 1α-mediated VEGF secretion and that IL-1β-stimulated ERK1/2 activation may be involved in this process.

Methoxychlor (MXC) is an organochlorine pesticide that increases the rate of ovarian atresia. To date, little is known about the mechanism by which MXC induces atresia. Because Bcl-2 (an antiapoptotic factor), Bax (a proapoptotic factor), gonadotropins, and estradiol are important regulators of atresia in the ovary, the purpose of this study was first to examine whether MXC-induced atresia occurred through alterations in Bcl-2 or Bax, and second, to examine the effect of MXC on gonadotropins, estradiol, and their receptors. CD-1 mice were dosed with 8–64 mg kg⁻¹ day⁻¹ MXC or vehicle (sesame oil). Ovaries were subjected to analysis of antral follicle numbers, Bcl-2, Bax, estrogen receptor, and follicle-stimulating hormone receptor levels. Blood was used to measure gonadotropins and estradiol. In some experiments, mice that overexpressed Bcl-2 or mice that were deficient in Bax were dosed with MXC or vehicle and their ovaries were analyzed for atresia. MXC caused a dose-dependent increase in the percentage of atretic antral follicles compared with controls at the 32 and 64 mg kg⁻¹ day⁻¹ doses of MXC. MXC treatment did not result in changes in Bcl-2 levels, but it did result in an increase in Bax levels in antral follicles. MXC treatment did not affect gonadotropin or estradiol levels, nor did it affect the levels of follicle-stimulating hormone or estrogen receptors. Mice that overexpressed Bcl-2 or mice that were deficient in Bax were protected from MXC-induced atresia. These data suggest that MXC induces atresia through direct effects on the Bax and Bcl-2 signaling pathways in the ovary.

This study evaluated the potential of slow-release GnRH agonist (deslorelin) implants to inhibit reproductive function in the male tammar wallaby. The specific aim was to measure the effects of graded dosages of deslorelin on testes size and plasma LH and testosterone concentrations. Adult male tammar wallabies were assigned to four groups (n = 6 per group) and received the following treatment: control, placebo implant; low dose, 5 mg deslorelin; medium dose, 10 mg; high dose, 20 mg. All dosages of deslorelin induced acute increases (P < 0.001) in plasma LH and testosterone concentrations within 2 h, with concentrations remaining elevated during the first 24 h but returning to pretreatment levels by Day 7. Thereafter, there was no evidence of a treatment-induced decline in plasma testosterone concentrations. There was no detectable difference in basal LH concentrations between treated and control animals, nor was there a significant change in testes width or length (P > 0.05). These results suggest that the male tammar wallaby is resistant to the contraceptive effects of chronic GnRH agonist treatment. Despite the maintenance of testosterone secretion, the majority of male tammars (10 of 17) failed to respond to a GnRH challenge with a release of LH between Days 186 and 197 of treatment. The failure of animals to respond to exogenous GnRH suggests a direct effect of deslorelin on the pituitary, resulting in a level of desensitization that was sufficient to inhibit a LH surge but insufficient to inhibit basal LH secretion. The variation between animals is believed to result from earlier recovery of some individuals, in particular those that received a lower dose, or individual resistance to the desensitization process.

When oocytes resume meiosis, chromosomes start to condense and Cdc2 kinase becomes activated. However, recent findings show that the chromosome condensation does not always correlate with the Cdc2 kinase activity in pig oocytes. The objectives of this study were to examine 1) the correlation between chromosome condensation and histone H3 phosphorylation at serine 10 (Ser10) during the meiotic maturation of pig oocytes and 2) the effects of protein phosphatase 1/2A (PP1/ PP2A) inhibitors on the chromosome condensation and the involvement of Cdc2 kinase, MAP kinase, and histone H3 kinase in this process. The phosphorylation of histone H3 (Ser10) was first detected in the clump of condensed chromosomes at the diakinesis stage and was maintained until metaphase II. The kinase assay showed that histone H3 kinase activity was low in oocytes at the germinal vesicle stage (GV) and increased at the diakinesis stage and that high activity was maintained until metaphase II. Treatment of GV-oocytes with okadaic acid (OA) or calyculin-A (CL-A), the PP1/PP2A inhibitors, induced rapid chromosome condensation with histone H3 (Ser10) phosphorylation after 2 h. Both histone H3 kinase and MAP kinase were activated in the treated oocytes, although Cdc2 kinase was not activated. In the oocytes treated with CL-A and the MEK inhibitor U0126, neither Cdc2 kinase nor MAP kinase were activated and no oocytes underwent germinal vesicle breakdown (GVBD), although histone H3 kinase was still activated and the chromosomes condensed with histone H3 (Ser10) phosphorylation. These results suggest that the phosphorylation of histone H3 (Ser10) occurs in condensed chromosomes during maturation in pig oocytes. Furthermore, the chromosome condensation is correlated with histone H3 kinase activity but not with Cdc2 kinase and MAP kinase activities.

Two-cell bovine embryos become arrested in development when exposed to a physiologically relevant heat shock. One of the major ultrastructural modifications caused by heat shock is translocation of organelles toward the center of the blastomere. The objective of the present study was to determine if heat- shock-induced movement of organelles is a result of cytoskeletal rearrangement. Two-cell bovine embryos were cultured at 38.5°C (homeothermic temperature of the cow), 41.0°C (physiologically relevant heat shock), or 43.0°C (severe heat shock) for 6 h in the presence of either vehicle, latrunculin B (a microfilament depolymerizer), rhizoxin (a microtubule depolymerizer), or paclitaxel (a microtubule stabilizer). Heat shock caused a rearrangement of actin-containing filaments as detected by staining with phalloidin. Moreover, latrunculin B reduced the heat-shock-induced movement of organelles at 41.0°C but not at 43.0°C. In contrast, movement of organelles caused by heat shock was inhibited by rhizoxin at both temperatures. Furthermore, rhizoxin, but not latrunculin B, reduced the swelling of mitochondria caused by heat shock. Paclitaxel, while causing major changes in ultrastructure, did not prevent the movement of organelles or mitochondrial swelling. It is concluded that heat shock disrupts microtubule and microfilaments in the two-cell bovine embryo and that these changes are responsible for movement of organelles away from the periphery. In addition, intact microtubules are a requirement for heat-shock-induced swelling of mitochondria. Differences in response to rhizoxin and paclitaxel are interpreted to mean that deformation of microtubules can occur through a mechanism independent of microtubule depolymerization.

The injection of male haploid germ cells, such as spermatozoa and round spermatids, into preactivated mouse oocytes can result in the development of viable embryos and offspring. However, it is not clear how the timing of intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) affects the production of offspring. We carried out ICSI and ROSI every 20 min for up to 4 h after the activation of mouse oocytes by Sr² and compared the late-stage development of ICSI- and ROSI- treated oocytes, including the formation of pronuclei, blastocyst formation, and offspring production. The rate of pronucleus formation (RPF) after carrying out ICSI started to decrease from >95% at 100 min following oocyte activation and declined to <20% by 180 min. In comparison, RPF by ROSI decreased gradually from >70% between 0 and 4 h after activation. The RPFs were closely correlated with blastocyst formation. Offspring production for both ICSI and ROSI decreased significantly when injections were conducted after 100 min, a time at which activated oocytes were in the early G1 stage of the cell cycle. These results suggest that spermatozoa and round spermatids have different potentials for inducing the formation of a male pronucleus in activated oocytes, but ICSI and ROSI are both subject to the same time constraint for the efficient production of offspring, which is determined by the cell cycle of the activated oocyte.

Postnatal uterine development involves differentiation and development of the endometrial glandular epithelium from the luminal epithelium as well as development of the mesenchyme into the endometrial stroma and myometrium. This period of development is critical because exposure of neonates to endocrine disruptors compromises reproductive cycles and pregnancy in the adult. However, the hormonal, cellular, and molecular mechanisms regulating postnatal uterine development remain largely unknown. In order to identify candidate genes and gene networks that regulate postnatal uterine development, uteri were collected from CD-1 outbred mice on postnatal days (PND) 3, 6, 9, 12, and 15, and gene expression profiling was conducted using Affymetrix mouse genome U74Av2 GeneChips in study 1. Of the approximately 12 000 genes analyzed, 9002 genes were expressed in the uterus and expression of 3012 genes increased or decreased 2-fold during uterine development. In study 2, the uterine epithelium was enzymatically separated from the stroma/myometrium on PNDs 3, 6, and 9, and gene expression profiling was conducted using CodeLink UniSet Mouse I Expression Bioarrays. Results from these two studies support the hypothesis that postnatal uterine development is a complex process involving overlapping positive and negative changes in uterine epithelial and stromal/myometrial gene expression. Candidate genes regulating uterine development encode secreted factors (Wnt5a, Wnt7a), transcription factors (Hoxa10, Hoxa11, Hoxd10, MSX-1), enzymes (matrix metalloproteinases, cathepsin, carbonic anhydrase), growth factors (IGF-II, IGF binding proteins), and components of the extracellular matrix (osteopontin) to name a few. The candidate genes and gene networks identified by transcriptional profiling provide an important foundation to discern and understand mechanisms regulating postnatal uterine morphogenesis.

Chemokines and chemokine receptors have been implicated as pivotal players in many physiological and pathological situations, but little is known about the expression and function of chemokines and chemokine receptors at the materno-fetal interface. In this study, we first analyzed the transcription of 18 chemokine receptors in first-trimester human trophoblast cells. Among these receptors, CXCR4 was found highly transcribed. We demonstrated afterward that both CXCR4 and CXCL12 (stromal cell-derived factor-1; SDF-1) were expressed in trophoblast cells. Primary cultured trophoblast cells were also found secreting CXCL12 spontaneously. To identify the functional role of CXCR4/CXCL12 in these cells, we treated trophoblast cells with recombinant human (rh)SDF-1α and analyzed the cell viability and signaling pathway. The results showed that rhSDF-1α increased the viability of trophoblast cells and the activation of extracellular signal-regulated kinases signaling pathway in vitro. Our findings suggest that first-trimester trophoblast cells express functional CXCR4/CXCL12, which may play an important role in early pregnancy such as stimulating trophoblast cell proliferation or differentiation in an autocrine manner.

The objective of the current study was to develop an ovine animal model for consistent study of uterine blood flow (UBF) changes during synchronized ovarian cycles regardless of season. Sheep were surgically bilaterally instrumented with uterine artery blood flow transducers and 5–7 days later implanted with a vaginal progesterone (P₄)-controlled internal drug-releasing device (CIDR; 0.3 g) for 7 days. On Day 6 of P₄, sheep were given two prostaglandin F_2α injections (7.5 mg i.m. 4 h apart). At CIDR removal, Experimental Day 0, zero (n = 9), 500 IU (n = 8), or 1000 IU (n = 7) eCG was injected i.m.; UBF was monitored continuously for 55–75 h. Jugular blood was sampled every 8 h to evaluate levels of P₄, estradiol-17β (E₂β) and luteinizing hormone (LH). The inhibitor of nitric oxide synthase, l- nitro-arginine methyl ester (l-NAME) was infused in a stepwise fashion unilaterally into one uterine artery at 48–50 h after 500 IU eCG and the effects on UBF were examined (n = 7). The zero-eCG group gradually increased UBF from a baseline of 17.4 ± 3.9 to 80.5 ± 1.1 ml/min. The 500-IU-eCG group increased UBF between 10 and 15 h from a baseline of 11 ± 3.3 to 83.3 ± 1.0 ml/min, whereas UBF for the 1000-IU-eCG group was higher (100.1 ± 1.7 ml/min) than that seen in either of the other groups. Plasma P₄ fell to baseline within 8 h of CIDR removal, while E₂β rose gradually in association with elevations in UBF. LH surges occurred between 32 and 56 h after CIDR removal and the LH surge occurred earlier in the 1000-IU-eCG group than the other two groups (P < 0.01). l-NAME infusion dose dependently reduced maximum levels of UBF ipsilaterally by 54.6% ± 6.2%, but contralaterally only by 27.4% ± 8.5%. Regardless of season, either dose of eCG will result in analogous UBF responses. During the follicular phase, elevations in UBF are in part locally controlled by the de novo production of nitric oxide.