Membrane motions of LH receptors following binding of hormone agonists are consistent with hormone-driven aggregation. It is increasingly apparent that G protein-coupled receptors, including the LH receptor, are engaged in dynamic interactions with one another and other membrane components. These interactions are governed, in part, by a number of factors including whether the receptor has bound ligand, whether the receptor is capable of transducing a hormone-mediated signal, and the nature of the membrane environment within which the receptor is found. Microscopic methods, including laser-optical techniques, are ideally suited to probe dynamic events on cell membranes and provide an opportunity to examine interactions between receptors and other membrane components on viable cells. We and others have used a variety of techniques, some of which are summarized below, to examine functional and nonfunctional LH receptors on viable cells and the membrane environment of these receptors during cell signaling events.

Estrogen signaling to GnRH neurons is critical for coordinating the preovulatory surge release of LH with follicular maturation. Until recently it was thought that estrogen signaled GnRH neurons only indirectly through numerous afferent systems. This minireview presents new evidence indicating that GnRH neurons are directly regulated by estradiol (E₂), primarily through estrogen receptor (ER)-β, and indirectly through E₂-sensitive neurons in the anteroventral periventricular (AVPV) region. The data described suggest that E₂ generally represses GnRH gene expression but that this repression is transiently overcome by indirect E₂-dependent signals relayed by AVPV neurons. We also present evidence that the AVPV neurons responsible for relaying E₂ signals to GnRH neurons are multifunctional gamma aminobutyric acid-ergic/glutamatergic/neuropeptidergic neurons.

In a previous article, we suggested that gp273, the ligand molecule for sperm-egg interaction in the bivalve mollusk Unio elongatulus has functional carbohydrate epitopes in common with a human zona pellucida glycoprotein, probably ZP3. We demonstrated that: 1) anti-gp273-purified immunoglobulin G (IgG), which recognizes a carbohydrate gp273 epitope including a Lewis^a-like structure, interacts with a zona pellucida protein; 2) human sperm specifically bind to gp273; and 3) binding is reversed by anti-gp273 IgG. In the present study, we confirm this suggestion by demonstrating that heat-solubilized zonae pellucidae reverse gp273-human sperm binding, that gp273-binding sites are restricted to the acrosomal region, and that gp273 induces the acrosome reaction in human sperm. We also demonstrated that gp273-binding sites on human sperm function as signaling receptors because exposure of spermatozoa to this glycoprotein results in significant stimulation of protein kinase C (PKC) activity. Because the PKC inhibitor, bisindolylmaleimide I, reverses both PKC activation and the acrosome reaction, this kinase is a key component of the signal transduction pathway activated by gp273 and leading to the exocytotic event.

The POU (Pit-Oct-Unc)-domain transcription factor, Oct-4, has become a useful marker of pluripotency in the mouse. It is found exclusively in mouse preimplantation-stage embryos after embryonic genome activation and is a characteristic of mouse embryonic stem (ES) cells, and its absence in knockout mice precludes inner cell mass (ICM) formation in blastocysts. Expression of Oct-4 has also been associated with pluripotency in primate cells. Here, we undertook a systematic study of Oct-4 expression in rhesus macaque preimplantation embryos produced by intracytoplasmic sperm injection and in ES cells before and after exposure to differentiating conditions in vitro. We also evaluated Oct-4 expression as a means of monitoring the extent of reprogramming following somatic cell nuclear transfer. Oct-4 was detected by reverse transcription-polymerase chain reaction and immunocytochemistry with a monoclonal antibody. Monkey pronuclear-stage zygotes and cleaving embryos up to the 8-cell stage showed no detectable Oct-4. Nuclear staining for Oct-4 first became obvious at the 16-cell stage, and a strong signal was observed in morula and compact morula stages. Both ICM and trophectodermal cell nuclei of monkey early blastocysts were positive for Oct-4. However, the signal was diminished in trophectodermal cells of expanded blastocysts, whereas expression remained high in ICM nuclei. Similar to the mouse, hatched monkey blastocysts showed strong Oct-4 expression in the ICM, with no detectable signal in the trophectoderm. Undifferentiated monkey ES cells derived from the ICM of in vitro-produced blastocysts expressed Oct-4, consistent with their pluripotent nature, whereas ES cell differentiation was associated with signal loss. Therefore, Oct-4 expression in the monkey, as in the mouse, provides a useful marker for pluripotency after activation of the embryonic genome. Finally, the observed lack or abnormal expression of Oct-4 in monkey nuclear transfer embryos suggests inadequate nuclear reprogramming.

Catecholestrogens are endogenous metabolites that have been shown to modulate granulosa, theca, and luteal cell function in some species. The present study was aimed at determining the possible role of these steroids on oocyte maturation. Cumulus-enclosed bovine oocytes were matured for 24 h, fertilized, and then cultured for 8 days. Whereas estradiol was without effect, addition of catecholestrogens (2-hydroxyestradiol, 4-hydroxyestradiol, and 2-methoxyestradiol [2-MOE2]) to the maturation medium did not affect the cleavage rate but was associated with a decrease in blastocyst production on Day 8. Although 2-MOE2 was also able to inhibit blastocyst formation when added during embryo culture, the effects were less pronounced than those seen when the steroid was added only during maturation. In agreement with the known ability of 2-MOE2 to bind tubulin at the colchicine site, marked alterations were observed in the spindle assembly of oocytes exposed to 2-MOE2 during maturation, which lead to gross chromosomal aberrations after fertilization and consequent developmental arrest at the morula stage. Moreover, that the blastocyst rate was not affected when meiosis was blocked with roscovitine during 2-MOE2 exposure is consistent with the idea that altered nuclear maturation is the cause of the low developmental competence. Because 2-MOE2 could be increased in follicular fluid in response to aryl hydrocarbon-receptor ligands, such as some environmental contaminants, our results show that abnormally high intraovarian levels of catecholestrogens could have a deleterious effect on oocyte maturation and early embryonic development arising from the alterations in the meiotic spindle.

The process of sperm capacitation is correlated with activation of a signal transduction pathway leading to protein tyrosine phosphorylation. Whereas phosphotyrosine expression is an essential prerequisite for fertilization, the proteins that are phosphorylated during capacitation have not yet been identified. In the present study, we observed that a major target of this signaling pathway is the molecular chaperone protein, heat shock protein (HSP)-86, a member of the HSP-90 family of HSPs. We used cross-immunoprecipitation experiments to confirm the tyrosine phosphorylation of HSP-86, a process that is not inhibited by the ansamycin antibiotic, geldanamycin. The general significance of these findings was confirmed by studies in which HSP-90 was also found to be tyrosine phosphorylated in human and rat spermatozoa when incubated under conditions that support capacitation. To our knowledge, these results represent the first report of a protein that undergoes tyrosine phosphorylation during mouse sperm capacitation and the first study implicating molecular chaperones in the processes by which mammalian spermatozoa gain the ability to fertilize the oocyte.

The Wilms tumor 1 (WT1) gene product may regulate the mullerian-inhibiting substance (MIS) gene, because mutations in WT1 can cause persistence of the mullerian duct in men. In the present study, we show by gel shift and chromatin immunoprecipitation assays that WT1 bound to a GC-rich sequence in the murine Mis promoter. Mutation in this site abolished WT1-mediated activation of the Mis promoter. The WT1, SRY box protein 9, and steroidogenic factor 1 could synergistically activate the Mis promoter, and at least two factors were necessary for minimal activation. The WT1 is an essential factor for activation of the Mis promoter; therefore, the persistence of the mullerian duct in patients with Denys-Drash syndrome may result from deregulation of the MIS gene.

Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/W ^v mutant mouse testis. In the present study, some of the W/W ^v mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/W ^v mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/W ^v spermatogonia into the seminiferous tubules of normal SCF (W/W ^v) or SCF mutant (Sl/Sl^d) mice demonstrated that the W/W ^v spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/W ^v testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/W ^v testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.

The Xenopus laevis egg vitelline envelope is composed of five glycoproteins (ZPA, ZPB, ZPC, ZPD, and ZPX). As shown previously, ZPC is the primary ligand for sperm binding to the egg envelope, and this binding involves the oligosaccharide moieties of the glycoprotein (Biol. Reprod., 62:766–774, 2000). To understand the molecular mechanism of sperm-egg envelope binding, we characterized the N-linked glycans of the vitelline envelope (VE) glycoproteins. The N-linked glycans of the VE were composed predominantly of a heterogeneous mixture of high-mannose (5-9) and neutral, complex oligosaccharides primarily derived from ZPC (the dominant glycoprotein). However, the ZPA N-linked glycans were composed of acidic-complex and high-mannose oligosaccharides, ZPX had only high-mannose oligosaccharides, and ZPB lacked N-linked oligosaccharides. The consensus sequence for N-linked glycosylation at the evolutionarily conserved residue N¹¹³ of the ZPC protein sequence was glycosylated solely with high-mannose oligosaccharides. This conserved glycosylation site may be of importance to the three-dimensional structure of the ZPC glycoproteins. One of the complex oligosaccharides of ZPC possessed terminal β-N-acetyl-glucosamine residues. The same ZPC oligosaccharide species isolated from the activated egg envelopes lacked terminal β-N-acetyl-glucosamine residues. We previously showed that the cortical granules contain β-N-acetyl-glucosaminidase (J. Exp. Zool., 235:335–340, 1985). We propose that an alteration in the oligosaccharide structure of ZPC by glucosaminidase released from the cortical granule reaction is responsible for the loss of sperm binding ligand activity at fertilization.

The plasma membrane of spermatozoa undergoes substantial remodeling during passage through the epididymal duct, principally because of changes in phospholipid composition, exchange of glycoproteins with epididymal fluid, and processing of existing membrane proteins. Here, we describe the interaction of an epididymal glycoprotein recognized by monoclonal antibody 2D6 with the plasma membrane of rat spermatozoa. Our goals have been to understand more about the mechanism of secretion of epididymal glycoproteins, how they interact with the sperm's plasma membrane, and their disposition within it. Reactivity to 2D6 monoclonal antibody was first detectable in principal cells in the distal caput epididymidis and as a soluble high-molecular-weight complex in the secreted fluid. It was not associated with membranous vesicles in the duct lumen. On cauda spermatozoa 2D6 monoclonal antibody recognized a 24-kDa glycoprotein (the subunit of a disulfide cross-linked homodimer of 48 kDa) that was present on the plasma membrane overlying the sperm tail. Binding of 2D6 to immature spermatozoa in vitro was cell-type specific but not species specific, and the antigen could only be extracted from cauda spermatozoa with detergents. Sequencing studies revealed that the 24-kDa glycoprotein was a member of the β-defensin superfamily of small pore-forming glycopeptides of which several others (ESP13.2, Bin1b, E-2, EP2, HE2) are found in the epididymis. This evidence suggests that some epididymal glycoproteins are secreted into the luminal fluid in a soluble form and bind to specific regions of the sperm's surface via hydrophobic interactions. Given the antimicrobial function of β-defensins, they have a putative role in protecting spermatozoa and the epididymis from bacterial infections.

Stampidine [2′,3′-didehydro-2′,3′-dideoxythymidine 5′-[p-bromophenyl methoxyalaninyl phosphate], a prodrug of stavudine (STV/d4T) with improved anti-HIV activity, is undergoing development as a novel nonspermicidal microbicide. Here, we report the stability of stampidine as a function of pH, preparation of a novel thermoreversible ovule formulation for mucosal delivery, its dissolution profile in synthetic vaginal fluid, and its mucosal toxicity potential as well as systemic absorption in the rabbit model. Stampidine was most stable under acidic conditions. Stampidine was solubilized in a thermoreversible ovule formulation composed of polyethylene glycol 400, polyethylene glycol fatty acid esters, and polysorbate 80. Does were exposed intravaginally for 14 days to an ovule formulation with and without 0.5%, 1%, or 2% stampidine corresponding to 1 × 10⁷- to 4 × 10⁷-fold higher than its in vitro anti-HIV IC₅₀ value. Vaginal tissues harvested on Day 15 were evaluated for mucosal toxicity and cellular inflammation. Additionally, does were exposed intravaginally to stampidine, and plasma collected at various time points was assayed by analytical HPLC for the prodrug and its bioactive metabolites. Stampidine did not cause mucosal inflammation. The vaginal irritation scores for 0.5–2% stampidine were within the acceptable range for clinical trials. The prodrug and its major metabolites were undetectable in the blood plasma. The marked stability of stampidine at acidic pH, its rapid spreadability, together with its lack of mucosal toxicity or systemic absorption of stampidine via a thermoreversible ovule may provide the foundation for its clinical development as an easy-to-use, safe, and effective broad-spectrum anti-HIV microbicide without contraceptive activity.

Mouse embryogenesis is dose sensitive to vascular endothelial growth factor-A (VEGF-A), and mouse embryos partially deficient in VEGF-A die in utero because of severe vascular defects. In this study, we investigate the possible causes that underlie this phenomenon. Although the development of vascular defects in VEGF-A-deficient embryos seems to suggest that endothelial differentiation depends on the presence of a sufficient level of VEGF-A, we were surprised to find that endothelial differentiation per se is insensitive to a significant loss of VEGF-A activity. Instead, the development of the multipotent mesenchymal cells, from which endothelial progenitors arise in the yolk sac, is most highly dependent on VEGF-A. As a result of VEGF-A deficiency, dramatically fewer multipotent mesenchymal cells are generated in the prospective yolk sac. However, among the small number of mesenchymal cells that do enter the prospective yolk sac, endothelial differentiation occurs at a normal frequency. In the embryo proper, vasculogenesis is initiated actively in spite of a significant VEGF-A deficiency, but the subsequent steps of vascular development are defective. We conclude that a full-level VEGF-A activity is not critical for endothelial specification but is important for two distinct processes before and after endothelial specification: the development of the yolk sac mesenchyme and angiogenic sprouting of blood vessels.

Mouse spermatozoa from the caudae epididymides could be freeze-dried without losing their ability to support normal development. Immature spermatozoa from the testes, in contrast, were damaged by freeze-drying. However, immature spermatozoa became resistant to freeze-drying after their treatment with diamide, which oxidizes free -SH groups. Conversely, epididymal spermatozoa were damaged by freeze-drying if first treated with dithiothreitol (DTT), which reduces -SS- bonds. The potential for freeze-drying damage seems likely to relate to the -SS- status of sperm proteins, in particular its protamines.

Pluripotent cell development in the mammalian embryo results in the sequential formation of several developmentally distinct populations, inner cell mass, primitive ectoderm, and the primordial germ lineage. Factors within medium conditioned by HepG2 cells (MEDII) have been implicated in the formation and maintenance of primitive ectoderm from inner cell mass cells both in vitro and in vivo. Here we demonstrate that MEDII, but not LIF, is able to support the maintenance and proliferation in culture of pluripotent cells derived from primitive ectoderm formed in vitro or during embryonic development. This distinguishes primitive ectoderm and inner cell mass (ICM) on the basis of cytokine responsiveness and validates the biological activity proposed for factors within MEDII in primitive ectoderm establishment and maintenance. Further, it potentially provides an alternative technology for the isolation of pluripotent cells from the mammalian embryo.

Spermatogonial transplantation provides a straightforward approach to quantify spermatogonial stem cells (SSCs). Because donor-derived spermatogenesis is regenerated in the form of distinct colonies, the number of functional SSCs can be obtained by simply counting the number of colonies established in recipient testes. However, this approach is legitimate only when one colony arises from one stem cell (one colony-one stem cell hypothesis). In this study, we evaluated the validity of this hypothesis. Two populations of donor cells were obtained from the testes of two transgenic mouse lines and mixed at a 1:1 ratio. Following transplantation of the cell mixture, donor-derived colonies were visualized and individually excised, and genomic DNA was extracted from each colony. Based on unique marker genes of the two transgenic lines, the genotype of the cells contained in a colony was examined by polymerase chain reaction. A colony was determined to be clonal when only one transgene was detected. The results showed that 100% and 90% of colonies were clonal when <5 and 19 colonies were formed per recipient testis, respectively. However, the clonality of colonies decreased as the colony number per recipient testis or the length of each colony increased. These results support the one colony-one stem cell hypothesis and demonstrate that spermatogonial transplantation provides a highly quantitative assay for SSCs; however, these conclusions are applicable under a defined transplantation condition.

When the Y chromosome of a Mus musculus domesticus male mouse (caught in Tirano, Italy) is placed on a C57BL/6J genetic background, approximately half of the XY (B6.Y^TIR) progeny develop into normal-appearing but infertile females. We have previously reported that the primary cause of infertility can be attributed to their oocytes. To identify the primary defect in the XY oocyte, we examined the onset and progress of meiotic prophase in the B6.Y^TIR fetal ovary. Using bromo-deoxyuridine incorporation and culture, we determined that the germ cells began to enter meiosis at the developmental ages and in numbers comparable to those in the control XX ovary. Furthermore, the meiotic prophase appeared to progress normally until the late zygotene stage. However, the oocytes that entered meiosis early in the XY ovary failed to complete the meiotic prophase. On the other hand, a considerable number of oocytes entered meiosis at late developmental stages and completed the meiotic prophase in the XY ovary. We propose that the timing of entry into meiosis and the XY chromosomal composition influence the survival of oocytes during meiotic prophase in the fetal ovary.

Cloned mammals with normal fertility have been produced by nuclear transfer. Thus, oocyte cytoplasm has the ability to convert differentiated somatic cell nuclei into a state that resembles the conditions that occur at fertilization (nuclear reprogramming). Despite the long-held assumption that reprogramming factors are present in mammalian oocytes, the molecular nature of these factors is not known. The present study demonstrates that the process of nuclear reprogramming is not directly regulated by maturation promoting factor or mitogen-activated protein kinase activity. The potential for nuclear-transferred oocytes to develop to the blastocyst stage was not different when somatic cells at the M phase were fused with oocytes activated with ionomycin and cycloheximide 1–5 h before (12%–22%) but was significantly decreased when oocytes were activated 6 h before (1%). Further molecular studies on the differences between oocytes with and without reprogramming potential are required and will be useful for the identification of reprogramming factors.

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6–9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.

We have suggested in a previous in vitro study that tumor necrosis factor-α (TNFα) plays a role in the initiation of luteolysis in cattle. The aim of the present study was to examine the influence of different doses of TNFα on the estrous cycle in cattle by observing the standing behavior and measuring peripheral concentrations of progesterone (P4) during the estrous cycle. Moreover, we evaluated the secretion of P4, oxytocin (OT), nitric oxide (NO), and luteolytic (prostaglandin F_2α [PGF_2α] and leukotriene C₄ [LTC₄]) and luteotropic (PGE₂) metabolites of arachidonic acid in peripheral blood plasma as parameters of TNFα actions. Mature Holstein/Polish black and white heifers (n = 36) were treated on Day 14 of the estrous cycle (Day 0 = estrus) by infusion into the aorta abdominalis of saline (n = 8), an analogue of PGF_2α (cloprostenol, 100 μg; n = 3) or saline with TNFα at doses of 0.1 (n = 3), 1 (n = 8), 10 (n = 8), 25 (n = 3), or 50 μg (n = 3) per animal. Peripheral blood samples were collected frequently before, during, and up to 4 h after TNFα treatment. After Day 15 of the estrous cycle, blood was collected once daily until Day 22 following the first estrus. Lower doses of TNFα (0.1 and 1 μg) decreased the P4 level during the estrous cycle and consequently resulted in shortening of the estrous cycle (18.8 ± 0.9 and 18.0 ± 0.7 days, respectively) compared with the control (22.3 ± 0.3 days, P < 0.05). One microgram of TNFα increased the PGF_2α (P < 0.001) and NO (P < 0.001) concentrations and decreased OT secretion (P < 0.01). Higher doses of TNFα (10, 25, 50 μg) stimulated synthesis of P4 (P < 0.001) and PGE₂ (P < 0.001), inhibited LTC₄ secreton (P < 0.05), and consequently resulted in prolongation of the estrous cycle (throughout 30 days, P < 0.05). Altogether, the results suggest that low concentrations of TNFα cause luteolysis, whereas high concentrations of TNFα activate corpus luteum function and prolong the estrous cycle in cattle.

Calcium plays roles in excitability, rhythm generation, and neurosecretion. Identifying channel subtypes that regulate calcium influx is thus important to understanding rhythmic GnRH secretion, which is a prerequisite for reproduction. Whole-cell voltage-clamp recordings were made from short-term dissociated GnRH adult ovariectomized (OVX) mice (n = 21) to identify channel subtypes that carry calcium current using selective channel blockers and voltage characteristics. Low-voltage activated (LVA) currents were not observed in 42 GnRH neurons tested, although most non-GnRH neurons (4/6) displayed LVA current. The L-type component of the high-voltage activated (HVA) calcium current was 25% ± 2%. The remaining HVA calcium current passed through N-type (27% ± 3%), P-type (15% ± 1%), Q-type (18% ± 3%), and R-type (15% ± 1%) channels. Because these data differ substantially from reports on cultured GnRH neurons, which may represent reproductively immature models, we also examined GnRH neurons from gonadal-intact young (Postnatal Days 4–10, n = 8 mice) mice. LVA currents were still rare (2/28) in young mice. Although the same HVA components were observed, the proportions were shifted toward significantly more L-type and less N-type current, suggesting a possible developmental shift in calcium currents in GnRH neurons. These data suggest that calcium channel subtypes in GnRH neurons prepared in the short term from brain slices differ substantially from those in long-term cultured GnRH models. These findings provide a vital foundation to examine the role of calcium channels in the secretory and rhythmic machinery of GnRH neurons.

The primary structure of mouse SVS I was determined by peptide sequencing and nucleotide sequencing of cloned cDNA. The precursor molecule consists of 820 amino acid residues, including a signal peptide of 24 residues, and the mature polypeptide chain of 91 kDa has one site for potential N-linked glycosylation. The SVS I is homologous with amiloride-binding protein 1 (ABP1), a diamine oxidase. However, it probably lacks enzymatic activity, because the cDNA codes for His instead of Tyr at the position of the active-site topaquinon. The SVS I monomer probably binds one molecule of copper, because the His residues coordinated by Cu(II) are conserved. The SVS I gene consists of five exons and is situated on mouse chromosome 6,B2.3. It is located in a region of 100 kilobases (kb) containing several genes with homology to SVS I, including the gene of ABP1 and two other proteins with homology to diamine oxidase. The locus is conserved on rat chromosome 4q24, but the homologous region on human chromosome 7q34-q36 solely contains ABP1. The other genes with homology to diamine oxidase were probably present in a progenitor of primates and rodents but were lost in the evolutionary lineage leading to humans—presumably during recombination between chromosomes. The estimated molecular mass of rat SVS I is 102 kDa (excluding glycosylation). The species difference in size of SVS I is caused by tandem repeats of 18 amino acid residues in the central part of the molecule: The mouse has seven repeats, and the rat has 12 repeats.

Complement C4b-binding protein (C4BP) is a plasma protein synthesized in the liver and plays a regulatory role in the host defense complement system. We have previously reported that mRNAs of the C4BP α chain (C4BPα) are expressed at significant levels in the guinea pig and mouse epididymis in an androgen-dependent manner. Here, we analyze the murine C4bpa gene and show that epididymal and liver C4BPα mRNAs are generated from a single-copy gene and that the epididymal C4BPα mRNAs are transcribed from novel transcription start sites located approximately 100 base pairs downstream from those used in the liver. Furthermore, in an immunohistochemical study using rabbit anti-mouse C4BP antiserum, we demonstrated that C4BP is localized in the stereocilia and Golgi apparatus of the epididymal epithelial cells and the surfaces of spermatozoa in the lumen in the region from the distal caput to the cauda but not in the proximal caput region. Indirect immunofluorescence of the isolated spermatozoa demonstrated that C4BP is localized preferentially on the head region of the spermatozoa, and immunoelectron microscopy located C4BP on the plasma membrane and the outer acrosomal membrane. These results indicate that epididymal C4BP is synthesized in the epithelial cells and secreted into the lumen in a region-restricted manner and is taken up to the sperm membranes on passage through the epididymis. Many epididymal proteins are secreted from the epithelial cells in a region-specific and androgen-dependent manner and are considered to contribute to sperm maturation. Our findings suggest a novel function of C4BP as one such epididymal secretory protein.

Germ cell transplantation is a technique that transfers donor testicular cells into recipient testes. A population of germ cells can colonize the recipient testis, initiate spermatogenesis, and produce sperm capable of fertilization. In the present study, a nonmosaic Klinefelter bull was used as a germ cell recipient. The donor cell suspension was introduced into the rete testis using ultrasound-guided puncture. A pulsatile administration of GnRH was performed to stimulate spermatogenesis. The molecular approach to detect donor cells was done by a quantitative polymerase chain reaction with allele discrimination based on a genetic mutation between donor and recipient. Therefore, a known genetic mutation, associated with coat-color phenotype, was used to calculate the ratio of donor to recipient cells in the biopsy specimens and ejaculates for 10 mo. After slaughtering, meiotic preparations were performed. The injected germ cells did not undergo spermatogenesis. Six months after germ cell transplantation, the donor cells were rejected, which indicates that the donor cells could not incorporate in the testis. The hormone stimulation showed that the testosterone-producing Leydig cells were functionally intact. Despite subfertility therapy, neither the recipient nor the donor cells underwent spermatogenesis. Therefore, nonmosaic Klinefelter bulls are not suitable as germ cell recipients. Future germ cell recipients in cattle could be mosaic Klinefelters, interspecies hybrids, bulls with Sertoli cell-only syndrome, or bulls with disrupted germ cell migration caused by RNA interference.

Glucocorticoids are known to influence many aspects of prenatal development. Three important regulators of glucocorticoid actions at the cellular level are the enzymes 11β-hydroxysteroid dehydrogenase type 1 (11βHSD-1), 11β-hydroxysteroid dehydrogenase type 2 (11βHSD-2), and glucocorticoid receptors (GR). The present study was conducted to determine the presence of these regulators in porcine placentae during early gestation (Days 24–40; term = 114 days) and to examine the influence of breed and uterine environment. Three pig models differing in uterine environment as reflected by embryonic survival from Days 24 to 40 were used: intact white cross-bred gilts (WC-INT); white cross-bred gilts that had been unilaterally hysterectomized-ovariectomized before puberty (WC-UHO); and intact Meishan gilts (ME). Porcine-specific partial cDNAs for 11βHSD-1 and 11βHSD-2 and a cRNA for GRα were developed and used to produce ³²P-labeled probes for Northern blot analyses. The 11βHSD dehydrogenase activity was measured in vitro at saturating concentrations of substrate and coenzyme. At Day 24 of gestation, 11βHSD-2 mRNA, dehydrogenase activity, and GR mRNA were present, but 11βHSD-1 mRNA was absent. All three mRNAs and dehydrogenase activity increased (P < 0.01) by Day 40. On Day 30, placental 11βHSD-2 mRNA was decreased (P = 0.03) by 47% in WC-UHO versus WC-INT. Placental 11βHSD dehydrogenase activity was 2-fold greater (P < 0.01) in ME versus WC-INT on Day 24 of gestation. These results demonstrate, to our knowledge for the first time, the presence of 11βHSD-1, 11βHSD-2, and GR mRNA as well as 11βHSD dehydrogenase activity in the porcine placenta during early pregnancy. Moreover, a role for glucocorticoids in porcine embryonic development is suggested.

Despite the enormous volume of research concerning the various effects of chemicals with endocrine-disrupting properties in fish, there is still very little evidence that endocrine disruption can adversely affect individual fertility and, hence, pose problems for the population. In the present study, guppies (Poecilia reticulata) were fed with the anti-androgenic fungicide vinclozolin at concentrations ranging from 1.8 to 180 mg/kg from 8–14 wk of age. Male sperm count and the intensity of his sexual display behavior were significantly reduced in treatment groups, which was in line with the results of previous studies. Here, we show further that these impairments translate into reduced fertility, measured as the size of the female's first clutch. Also, this reduced fertility was correlated to the male sperm count, but not to the intensity of the male sexual display. Finally, by crossing exposed with unexposed animals, we show that the adverse effect of vinclozolin on reproduction is mediated through the male alone.

Toll-like receptor 4 (TLR-4) is a critical mediator of the cellular response to lipopolysaccharide. Our purpose was to examine the role of TLR-4 in parturition and in the regulation of expression of prostaglandin synthase (cyclooxygenase [COX]-1 and COX-2) and 15-hydroxyprostaglandin dehydrogenase (PGDH) following exposure to heat-killed Escherichia coli (HKE) in pregnant mice. Inbred TLR-4-mutant C3H/HeJ mice and inbred normal C3HeB/FeJ mice on Day 14.5 of a 19- to 20-day gestation received intrauterine injection of either HKE or sterile vehicle (PBS). Preterm or term delivery was recorded for these animals. Tissues (myometrium, decidual caps, placentas, fetal membranes, and fetuses) were collected after injection of sterile vehicle or 5 × 10⁹ HKE bacteria (n = 5 mice per strain per treatment per time point). The COX-1, COX-2, and PGDH gene expression was determined by semiquantitative reverse transcription-polymerase chain reaction. We found that 5 × 10⁹ HKE induced preterm delivery in 100% of TLR-4-normal mice but in 0% of TLR-4-mutant mice. The HKE exposure up-regulated expression of COX-2, but not of COX-1, in maternal tissues in both mouse strains. The prostaglandin-catabolizing enzyme PGDH was down-regulated in myometrium, fetal membranes, and fetuses in control mice, but no change was observed in TLR-4-mutant mice after HKE treatment. These results demonstrate that a functional TLR-4 is essential for HKE-induced preterm labor and PGDH down-regulation but is not essential for HKE-induced COX-2 gene up-regulation. The TLR-4 may mediate bacterially induced preterm labor via regulation of prostaglandin degradation rather than prostaglandin synthesis.

Di-(2-ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been shown to cause reproductive toxicity in both developing and adult animals. In this study, we used organ cultures of fetal and neonatal rat testes to assess the in vitro effect of MEHP on seminiferous cord formation in Embryonic Day 13 (E13) testes and on the development of E18 and Postnatal Day 3 (P3) testes. Interestingly, MEHP had no effect on cord formation in the organ cultures of E13 testes, indicating that it has no effect on sexual differentiation of the indifferent gonad to testis. Consistently, the expression of a Sertoli cell-specific protein, mullerian inhibiting substance (MIS), or the number of gonocytes did not change in E13 testes after MEHP treatment. In contrast, MEHP decreased the levels of MIS and GATA-4 proteins in Sertoli cells and impaired Sertoli cell proliferation in the organ cultures of E18 and P3 testes. These results suggest that MEHP negatively influences proliferation and differentiation of Sertoli cells in both fetal and neonatal testes. In addition, MEHP treatment did not alter the number of gonocytes in E18 testes, whereas the number of gonocytes in P3 testes decreased in a dose-dependent manner, apparently due to enhanced apoptosis. These results suggest that MEHP adversely affects the gonocytes, which are mitotically active and undergoing migration and differentiation in neonatal testes, but it has no effect on fetal gonocytes that are mitotically quiescent.

In recent years, much knowledge about the functions of defined genes in spermatogenesis has been gained by making use of mouse transgenic and gene knockout models. Single null mutations in mouse genes encoding four male germ cell proteins, transition protein 2 (Tnp-2), proacrosin (Acr), histone H1.1 (H1.1), and histone H1t (H1t), have been generated and analyzed. Tnp-2 is believed to participate in the removal of the nuclear histones and initial condensation of the spermatid nucleus. Proacrosin is an acrosomal protease synthesized as a proenzyme and activated into acrosin during the acrosome reaction. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues and germ cells during the S-phase of the cell cycle. The histone gene H1t is expressed exclusively in spermatocytes and may have a function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines. Male mutant mice lacking any of these proteins show no apparent defects in spermatogenesis or fertility. To examine the synergistic effects of these proteins in spermatogenesis and during fertilization, two lines of triple null mice (Tnp-2^−/−/Acr^−/−/H1.1^−/− and Tnp-2^−/−/Acr^−/−/H1t^−/−) were established. Both lines are fertile and show normal sperm parameters, which clearly demonstrate the functional redundancy of these proteins in male mouse fertility. However, sperm only deficient for Acr (Acr^−/−) are able to compete significantly with sperm from triple knockout mice Tnp-2^−/−/Acr^−/−/H1.1^−/− (70.7% vs. 29.3%) but not with sperm from triple knockout mice Tnp-2^−/−/Acr^−/−/H1t^−/− (53.6% vs. 46.4%). These results are consistent with a model that suggests that some sperm proteins play a role during sperm competition.

Expression of Kit ligand (KL) mRNA is induced in primary prepuberal Sertoli cells by FSH and by other agents that increase cAMP levels. The cAMP effect is exerted at the transcriptional level and appears to be cell type specific, since it is not observed in other KL-expressing primary cells or cell lines. Deletion analysis of the 5′-flanking region of the mouse KL gene shows that the proximal promoter sequence between −88 and 8 from the transcriptional start site is necessary and sufficient to obtain the full cAMP responsiveness of the promoter in primary mouse Sertoli cells. In the −88/ 8 promoter region, several cis-acting elements play a role in cAMP response. The −88/−56 sequence is necessary for full induction of the gene, since its removal causes a drastic decrease in cAMP responsiveness; however, cAMP-stimulated expression is still observed with the minimal promoter region between −55 and 8. A more detailed mutational analysis of the minimal promoter region shows that mutations in the canonical TATA box sequence and in an immediately downstream GC-rich element completely abolish cAMP responsiveness. DNA-binding experiments show that transcription factor Sp1 binds to the −88/−56 fragment of the KL proximal promoter in both control and cAMP-stimulated cells, whereas a new cAMP-induced complex is observed when the −55/ 8 minimal promoter region is used as probe. The canonical TATA box sequence is essential for formation of the latter complex. We also show that the binding of an unknown nuclear factor (different from Sp1, Egr-1, Rnf6, and AP-2) to a GC-rich element between −19 and 8 increases after cAMP treatment, and this effect seems to be specific of primary Sertoli cells. Thus, cAMP-induced transcription from the KL gene promoter in primary mouse Sertoli cells is mediated by a complex interaction among a Sp1-binding region, factors recognizing the canonical TATA box sequence, and a not yet identified cAMP-induced factor binding a GC-rich sequence just downstream from it.

Spermatogonia in the mouse testis arise from early postnatal gonocytes that are derived from primordial germ cells (PGCs) during embryonic development. The proliferation, self-renewal, and differentiation of spermatogonial stem cells provide the basis for the continuing integrity of spermatogenesis. We previously reported that Pin1-deficient embryos had a profoundly reduced number of PGCs and that Pin1 was critical to ensure appropriate proliferation of PGCs. The current investigation aimed to elucidate the function of Pin1 in postnatal germ cell development by analyzing spermatogenesis in adult Pin1^−/− mice. Although Pin1 was ubiquitously expressed in the adult testis, we found it to be most highly expressed in spermatogonia and Sertoli cells. Correspondingly, we show here that Pin1 plays an essential role in maintaining spermatogonia in the adult testis. Germ cells in postnatal Pin1^−/− testis were able to initiate and complete spermatogenesis, culminated by production of mature spermatozoa. However, there was a progressive and age-dependent degeneration of the spermatogenic cells in Pin1^−/− testis that led to complete germ cell loss by 14 mo of age. This depletion of germ cells was not due to increased cell apoptosis. Rather, detailed analysis of the seminiferous tubules using a germ cell-specific marker revealed that depletion of spermatogonia was the first step in the degenerative process and led to disruption of spermatogenesis, which resulted in eventual tubule degeneration. These results reveal that the presence of Pin1 is required to regulate proliferation and/or cell fate of undifferentiated spermatogonia in the adult mouse testis.

We have previously demonstrated that activin is likely an ovarian mediator of pituitary gonadotropin(s) and local epidermal growth factor in their stimulating oocyte maturation and maturational competence in the zebrafish. However, the downstream events controlled by activin remain unknown. One possible mechanism is that activin may directly work on the oocytes to promote the development of oocyte maturational competence. To substantiate this hypothesis, we performed the present study to demonstrate the expression of the activin system in different compartments of zebrafish follicles, namely, the follicle cells and oocytes. The proteins examined include activin subunits (βA and βB), activin-binding protein (follistatin), activin type II receptors (type IIA and IIB), the type I activin receptor-like kinases (ALK1-like, ALK2-like, and ALK4-like), and the intracellular activin signaling molecules (Smad2, Smad3, Smad4, and Smad7). The results showed that the entire activin signaling system is expressed by the full-grown immature zebrafish oocytes (∼0.65 mm in diameter), including ALK4-like (ActRIB), ALK2-like (ActRIA), ActRIIA, ActRIIB, Smad2, Smad3, Smad4, and Smad7, therefore supporting our hypothesis that the oocytes are one of the direct targets of activin actions in the zebrafish ovary. In contrast, activin itself (βA and βB) and ALK1-like type I receptor are predominantly expressed in the follicle cells surrounding the oocytes. Interestingly, although follistatin is expressed in both the follicle cells and oocytes, its level of expression is significantly higher in the oocytes than the follicle cells, implying that follistatin may serve as a signal from the oocytes to modulate the activity of activin produced by the follicle cells. Taken together, the present study provides convincing evidence that although all members of the activin system are expressed in the whole follicle, they exhibit distinct spatial patterns of expression among different compartments of the follicle. It is likely that activin works directly on the oocytes in a paracrine manner to promote oocyte maturation and maturational competence. On the other hand, instead of being controlled passively by the follicle cells, the oocytes may actively participate in the regulation of follicle development by releasing various modulating molecules such as follistatin.

Human embryonic stem (hES) cells have been traditionally cultured on primary mouse embryonic fibroblasts (PMEFs). However, though STO cells have some advantages over PMEFs and human embryonic fibroblasts (hEFs) as feeder cells, they have never been used as feeder cells to establish hES cell lines. In this study, three hES cell lines (Miz-hES1, Miz-hES2, and Miz-hES3) were established from inner cell masses (ICM), using STO as feeder cells. The three hES cell lines had normal karyotypes and expressed high levels of alkaline phosphatase (AP), cell surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), and transcription factor Oct-4. After culture on STO cells for 2 yr, hES cells maintained the potential to form derivatives of all three embryonic germ layers. Our results show that STO feeder cells have the potential to support the establishment and maintenance of hES cell lines. In addition, our results suggest that laminin may play an important role in maintaining the undifferentiated proliferation of hES cells.

Enkephalin appears to exert an inhibitory action on LH secretion, but whether testosterone regulates enkephalin gene expression is unknown. This study tested the hypothesis that testosterone and/or season modulate preproenkephalin mRNA expression in specific areas of the hypothalamus. Romney Marsh rams were castrated (wethers) either during the breeding season or nonbreeding season and received intramuscular injections of either oil or testosterone propionate (five/group). Blood samples were taken for the assay of plasma LH and testosterone. Preproenkephalin mRNA expression was quantified in hypothalamic sections by in situ hybridization. Mean plasma LH concentrations were reduced and the interpulse interval for LH pulses was greater in testosterone propionate-treated wethers compared with oil-treated wethers, with no change in LH pulse amplitude. Testosterone propionate treatment reduced proenkephalin expression in the diagonal band of Broca, the caudal preoptic area, and the bed nucleus of the stria terminalis. Seasonal differences in proenkephalin expression were observed in the bed nucleus of the stria terminalis, lateral septum, periventricular nucleus, and paraventricular nucleus. No differences were observed between treatments in seven other regions examined. We conclude that testosterone and season regulate proenkephalin mRNA levels in the preoptic area/hypothalamus in the ram in a region-specific manner.

Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3β-hydroxysteroid dehydrogenase (3β-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3β-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3β-HSD. In examining follicles of 3–5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to imply different mechanisms of initiation and regulation.

Mammalian sperm chromatin is bound by protamines into highly condensed toroids with approximately 50 kilobases (kb) of DNA. It is also organized into loop domains of about the same size that are attached at their bases to the proteinaceous nuclear matrix. In this work, we test our model that each sperm DNA-loop domain is condensed into a single protamine toroid. Our model predicts that the protamine toroids are linked by chromatin that is more sensitive to nucleases than the DNA within the toroids. To test this model, we treated hamster sperm nuclei with DNase I and found that the sperm chromatin was digested into fragments with an average size of about 50 kb, by pulse-field gel electrophoresis (PFGE). Surprisingly, we also found that spermatozoa treated with 0.25% Triton X-100 (TX) and 20 mM MgCl₂ overnight resulted in the same type of degradation, suggesting that sperm nuclei have a mechanism for digesting their own DNA at the bases of the loop domains. We extracted the nuclei with 2 M NaCl and 10 mM dithiothreitol (DTT) to make nuclear halos. Nuclear matrices prepared from DNase I-treated spermatozoa had no DNA attached, suggesting that DNase I digested the DNA at the bases of the loop domains. TX-treated spermatozoa still had their entire DNA associated with the nuclear matrix, even though the DNA was digested into 50-kb fragments as revealed by PFGE. The data support our donut-loop model for sperm chromatin structure and suggest a functional role for this type of organization in that sperm can digest its own DNA at the sites of attachment to the nuclear matrix.

The present study aimed to test the generally accepted view that a follicular wave starts with follicles newly recruited from the population smaller than 3 mm, which later compete for dominance. According to this view, subordinate follicles are expected to be too atretic to join the next follicular wave. Ten cows were ovariectomized shortly prior to the LH surge, thus around the start of the first follicular wave of the cycle. Per cow, on average, 14.4 follicles of ≥3 mm were dissected. Follicular health was determined on the basis of four parameters: 1) judgment of the degree of atresia by stereomicroscope, 2) incidence of apoptotic nuclei among the granulosa cells, 3) estradiol and progesterone concentrations, and 4) insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs)-2, -4, and -5 concentrations in the follicular fluid. In addition to the preovulatory follicle, 3.1 other follicles, mainly sized 3–4.5 mm, were found to be healthy based on the proportion of apoptotic nuclei, and concentrations of estradiol/progesterone, and IGFBPs. The ability of these follicles to respond with growth on the preovulatory and periovulatory FSH surges was supported by a comparison to the follicular population of four cows 31–68 h after the LH surge. The present results point to an alteration of the view on the follicular wave. The larger follicles during the first days of the follicular wave are, in general, derived from follicles that also joined the previous wave. A portion of these growing follicles are estradiol active and compete for dominance. Other growing follicles lack estradiol production and are probably derived from rather atretic follicles. The first newly recruited follicles do not reach the size of 3 mm before 31 h after the preovulatory FSH surge. At that time, the larger follicles are already competing for dominance.

In vitro use of arresters of meiosis could improve cytoplasmic maturation of immature oocytes by controlling the period of prophase I. Phosphodiesterases (PDE) are responsible for the breakdown and concomitant inactivation of the cyclic nucleotides cAMP and cGMP and are implicated in the regulation of oocyte meiotic maturation. Selective inhibitors of phosphodiesterase type 3 (PDE3) prevent meiotic resumption of mammalian oocytes. This study evaluated the impact of meiosis arrest by PDE3 inhibitor, Org 9935, on developmental competence of geminal vesicle (GV)-stage oocytes from small antral follicles. Cumulus-oocyte complexes (COC), retrieved from antral follicles 24 h after eCG exposure and cultured in the presence of PDE3 inhibitor (10 μM) for an additional 24 h, remained arrested in the meiotic prophase. The GV configuration of oocytes before and after the arrest by PDE3 inhibitor was examined. After the period of meiosis arrest, a significantly increased proportion of oocytes had acquired a nucleolus surrounded by a condensed chromatin rim at the GV, which is a morphological correlate of transcriptional repression. Removal of inhibitor resulted in 90.6% ± 8.3% of oocytes with the first polar body extruded. Fertilization was significantly improved in oocytes that had been arrested compared with oocytes collected 24 h after eCG and undergoing in vitro maturation immediately. Embryonic preimplantation and live offspring rates of arrested oocytes were higher, although not significantly, than those of nonarrested oocytes. These results suggest that a temporal block of meiosis by PDE3 inhibitor promotes developmental competence of mice oocytes retrieved from small antral follicles.

In the bovine corpus luteum (CL) phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in response to prostaglandin F_2α (PGF_2α) is correlated with the secretion of oxytocin. The present study was conducted to 1) examine the intracellular translocation characteristics of wild-type and mutant forms of a green fluorescent protein (GFP)-conjugated MARCKS (MARCKS-GFP) after PGF_2α treatment and 2) evaluate PGF_2α-induced temporal changes in MARCKS-GFP and actin cortex associated with exocytosis of oxytocin. In experiment 1, cells of the bovine CL were cultured on coverslips overnight. Then, wild-type and mutant MARCKS-GFP constructs were transfected separately into cells and expression was detected through fluorescence microscopy. Forty-eight hours after transfection, cells were treated with vehicle, PGF_2α (56 nM), or a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA], 1 μM). Treatment of cells expressing wild-type MARCKS-GFP with PGF_2α and TPA resulted in translocation of MARCKS from the plasma membrane to the cytoplasm within 2.5 min. Phosphorylation mutant MARCKS-GFP (m3) protein was localized on the plasma membrane, and treatments did not cause its translocation to the cytoplasm. Myristoylation mutant MARCKS-GFP (G2A) was observed solely in the cytoplasm, and no changes were detected in the intracellular location of this mutant MARCKS after treatment. In experiment 2, luteal cells were transfected with one of the three MARCKS-GFP constructs. Cells were then fixed and probed sequentially for oxytocin and filamentous actin. Results revealed that only wild-type MARCKS-GFP transfected large luteal cells contained advanced signs of exocytosis (peripheral movement of oxytocin vesicles; shorter actin filaments) with translocation of MARCKS-GFP from membrane to cytoplasm in response to PGF_2α treatment. These data demonstrate that phosphorylation of membrane-bound MARCKS protein is requisite for exocytosis of oxytocin to occur in bovine large luteal cells.

To better understand the differences in cytoskeletal organization between in vivo (IVO) and in vitro (IVM) matured oocytes, we analyzed remodeling of the centrosome-microtubule complex in IVO and IVM mouse oocytes. Fluorescence imaging revealed dramatic differences in meiotic spindle assembly and organization between these two populations. Metaphase spindles at both meiosis I (M-I) and meiosis II (M-II) in IVO oocytes were compact, displayed focused spindle poles with distinct γ-tubulin foci, and were composed of acetylated microtubules. In contrast, IVM oocytes exhibited barrel-shaped spindles with fewer acetylated microtubules and γ-tubulin diffusely distributed throughout the spindle proper. With respect to meiotic progression, IVO oocytes were more synchronous in the rate and extent of anaphase to telophase of M-I and first polar body emission than were IVM counterparts. Furthermore, IVO oocytes showed a twofold increase in cytoplasmic microtubule organizing centers (MTOCs), and constitutive MTOC proteins (γ-tubulin and pericentrin) were excluded from the first polar body. Inclusion of MTOC constitutive proteins in the polar body and diminished number of cytoplasmic MTOCs was observed in IVM oocytes. These findings were corroborated in IVO oocytes obtained from naturally ovulated and spontaneously cycling mice and highlight a fundamental distinction in the spatial and temporal regulation of microtubule dynamics between IVO and IVM oocytes

Exposure of cultured preimplantation embryos to temperatures similar to those experienced by heat-stressed cows inhibits subsequent development. In this study, the effects of heat shock on the ultrastructure of two-cell bovine embryos were examined to determine mechanisms for inhibition of development. Two-cell embryos produced in vitro were harvested at ∼28 h postinsemination and cultured for 6 h at one of three temperatures: 38.5°C (cow body temperature), 41.0°C (characteristic temperature for heat-stressed cows), or 43.0°C (severe heat shock). Ultrastructural examinations revealed that both heat shocks resulted in the movement of organelles towards the center of the blastomere. In addition, heat shock increased the percentage of mitochondria exhibiting a swollen morphology. Distance between the membranes comprising the nuclear envelope was increased but only when embryos were treated at 43.0°C. To determine whether ultrastructural responses to heat shock in culture were similar for embryos produced in vitro and in vivo, two-cell embryos were collected from superovulated Angus cows 48 h postinsemination and treated ex vivo for 6 h at 38.5°C or 41.0°C. Again, heat shock caused an increase in number of swollen mitochondria and movement of organelles away from the periphery of the blastomere. Exposure of two-cell bovine embryos to physiologically relevant elevated temperatures causes disruption in ultrastructural morphology that is inimical to development. The observation that overall morphology and response to heat was similar for embryos produced in vitro and in vivo implies that the former can be a good model for understanding embryonic responses to heat shock.

Angiogenesis is involved in the local mechanisms that regulate follicular development and ovulation. Recently, the angiopoietin (ANPT)-Tie system has been shown to be required to regulate angiogenesis and blood vessel regression. Expression of the ANPT-Tie system in the cyclic ovary suggests that the relative changes in the expression of ANPT-1 and ANPT-2 influence the stability of ovarian blood vessels. In this study, we investigated 1) the mRNA expression for ANPT-1, ANPT-2, and endothelial cell-specific receptors Tie1 and Tie2 in the theca interna (TI) of the bovine developing, mature, and atretic follicles by using a semiquantitative reverse transcription polymerase chain reaction assay and 2) the effect of ANPT on the secretion of steroid hormones from bovine preovulatory follicles in vitro using a microdialysis system (MDS) implanted in the thecal layer. Bovine follicles were classified as developing, mature, and atretic according to size, follicular fluid content of estradiol (E₂) and progesterone (P₄), and characteristics of granulosa cells (GCs). Both ANPT and Tie mRNA were expressed in the TI, whereas GCs expressed ANPT mRNA only. The expression of ANPT-2 mRNA was decreased in the mature follicles. This decrease resulted in a decrease in the ANPT-2:ANPT-1 ratio (an index of instability of blood vessels), indicating that the blood vessels became more stable or mature. The early atretic follicles showed a higher ANPT-2:ANPT-1 ratio and higher Tie2 mRNA expression than did other follicles at healthy or later atretic stages. This finding may imply that blood vessels become unstable at the initial stage of follicular atresia. In both mid and late atretic follicles, Tie2 mRNA expression dramatically decreased, indicating a disruption of the ANPT-Tie system. In the MDS experiment, an infusion of ANPT-1 or ANPT-2 increased P₄ release, whereas both ANPTs inhibited the release of androstenedione. ANPT-1 also increased E₂ release. These results showed that the mRNA expression for ANPT-1, ANPT-2, Tie1, and Tie2 changes during follicular development, maturation, and atresia in bovine follicles and that ANPTs affect steroidogenesis in the preovulatory follicle. The results suggest that the ANPT-Tie system is involved the structural (angiogenesis) and secretory changes that occur during follicular development and atresia.

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by the differentiation of a transient population of germ cells called gonocytes found in the center of the seminiferous tubule. The fate of gonocytes depends upon these cells resuming mitosis and developing the capacity to migrate from the center of the seminiferous tubule to the basement membrane. This process begins approximately Day 3 postpartum in the mouse, and by Day 6 postpartum differentiated type A spermatogonia first appear. It is essential for continual spermatogenesis in adults that some gonocytes differentiate into spermatogonial stem cells, which give rise to all differentiating germ cells in the testis, during this neonatal period. The presence of spermatogonial stem cells in a population of cells can be assessed with the use of the spermatogonial stem cell transplantation technique. Using this assay, we found that germ cells from the testis of Day 0–3 mouse pups can colonize recipient testes but do not proliferate and establish donor-derived spermatogenesis. However, germ cells from testes of Day 4–5 postpartum mice colonize recipient testes and generate large areas of donor-derived spermatogenesis. Likewise, germ cells from Day 10, 12, and 28 postpartum animals and adult animals colonize and establish donor-derived spermatogenesis, but a dramatic reduction in the number of colonies and the extent of colonization occurs from germ cell donors Days 12–28 postpartum that continues in adult donors. These results suggest spermatogonial stem cells are not present or not capable of initiating donor-derived spermatogenesis until Days 3–4 postpartum. The analysis of germ cell development during this time frame of development and spermatogonial stem cell transplantation provides a unique system to investigate the establishment of the stem cell niche within the mouse testis.

To further develop defined conditions for in vitro fertilization (IVF) and in vitro culture (IVC) of in vitro-matured porcine oocytes, we evaluated the effects of theophylline, adenosine, and cysteine in a chemically defined medium during IVF. Viability to full term of in vitro-produced blastocysts after IVF and IVC in chemically defined medium was also investigated by embryo transfer to recipients. A chemically defined medium, porcine gamate medium (PGM), was modified from porcine zygote medium (PZM-4), which was previously established. PGM was used as a basal medium for IVF and PZM-4 was for the culture of presumptive zygotes. Addition of 2.5 mM theophylline to PGM significantly increased the percentage of male pronuclear formation compared with controls (no addition). Addition of 1 μM adenosine to PGM supplemented either with or without 2.5 mM theophylline significantly reduced the number of penetrated spermatozoa compared with controls (no addition of adenosine). Supplementation with 0.2 μM cysteine in PGM containing both 2.5 mM theophylline and 1 μM adenosine further increased the percentage of development to the blastocyst stage, compared with no supplementation of cysteine, but there was no difference in fertilization parameters, such as monospermy and pronuclear formation, regardless of presence or absence of theophylline and adenosine. When Day 5 blastocysts were transferred into four recipients (20–25 blastocysts per recipient), all recipients became pregnant and farrowed a total of 21 live piglets. The present results clearly demonstrate that porcine blastocysts can be produced by IVF and IVC in chemically defined media and that they can develop to full term after embryo transfer.

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4°C. Samples frozen without cryoprotection were maintained at −196°C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then be stored at 4°C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.

In male salmonids, the age of maturation varies from 1 to 6 years and is influenced by growth during critical periods of the life cycle. The endocrine mechanisms controlling spermatogenesis and how growth affects this process are poorly understood. Recent research has indicated that gonadotropins, 11-ketotestosterone, and insulin-like growth factor I play roles in spermatogenesis in fish. To expand our understanding of the roles of these endocrine factors in onset of puberty, male spring chinook salmon (Oncorhynchus tshawytscha) were sampled at monthly intervals 14 mo prior to spermiation. This sampling regime encompassed two hypothesized critical periods when growth influences the initiation and completion of puberty for this species. Approximately 80% of the males matured during the experimental period, at age 2 in September 1999. An initial decline in the ratio of primary A to transitional spermatogonia was observed from July to December 1998, and during this period plasma levels of 11-ketotestosterone and pituitary levels of FSH increased. From January 1999 onward, males with low plasma 11-ketotestosterone levels (<1 ng/ml) had low pituitary and plasma FSH levels and no advanced development of germ cells. Conversely, from January through September 1999, males with high plasma 11-ketotestosterone levels (>1 ng/ml) had testes with progressively more advanced germ cell stages along with elevated pituitary and plasma FSH. Plasma levels of insulin-like growth factor I increased during maturation. These data provide the first physiological evidence for activation of the pituitary-testis axis during the fall critical period when maturation is initiated for the following year.