Animal spermatids and primary oocytes initially have typical centrosomes comprising pairs of centrioles and pericentriolar fibrous centrosomal proteins. These somatic cell–like centrosomes are partially or completely degenerated during gametogenesis. Centrosome reduction during spermiogenesis comprises attenuation of microtubule nucleation function, loss of pericentriolar material, and centriole degeneration. Centrosome reduction during oogenesis is due to complete degeneration of centrioles, which leads to dispersal of the pericentriolar centrosomal proteins, loss of replicating capacity of the spindle poles, and switching to acentrosomal mode of spindle organization. Oocyte centrosome reduction plays an important role in preventing parthenogenetic embryogenesis and balancing centrosome number in the embryonic cells.

The mouse USP8/mUBPy gene codifies a deubiquitinating enzyme expressed preferentially in testis and brain. While the ubiquitin-specific processing proteases (UBPs) are known to be important for the early development in invertebrate organisms, their specific functions remain still unclear in mammals. Using specific antibodies, raised against a recombinant mUBPy protein, we studied mUBPy in mouse testis. The mUBPy is expressed exclusively by the germ cell component and is maintained in epididymal spermatozoa. The enzyme is functionally active, being able to detach ubiquitin moieties from endogenous protein substrates. Protein interaction assays showed that sperm UBPy interacts with MSJ-1, the sperm-specific DnaJ protein evolutionarily conserved for spermiogenesis. Immunocytochemistry revealed that mUBPy shares with MSJ-1 the intracellular localization during spermatid cell differentiation; intriguingly, we show here that the proteasomes also locate in mUBPy/MSJ-1-positive sites, such as the cytoplasmic surface of the developing acrosome and the centrosomal region. These colocalization sites are maintained in epididymal spermatozoa. The demonstration of a protein interaction between a deubiquitinating enzyme and a molecular chaperone and the documentation on the proteasomes in both differentiating and mature mouse male germ cells suggest that members of the chaperone and ubiquitin/proteasome systems could cooperate in the fine control of protein quality to yield functional spermatozoa.

Mammalian testicular spermatozoa are immotile, thus, to reach the oocyte, they need to acquire swimming ability under the control of different factors acting during the sperm transit through the epididymis and the female genital tract. Although bicarbonate is known to physiologically increase motility by stimulating soluble adenylate cyclase (sAC) activity of mammalian spermatozoa, no extensive studies in human sperm have been performed yet to elucidate the additional molecular mechanisms involved. In this light, we investigated the effect of in vitro addition of bicarbonate to human spermatozoa on the main intracellular signaling pathways involved in regulation of motility, namely, intracellular cAMP production and protein tyrosine phosphorylation. Bicarbonate effects were compared with those of the phosphatidyl-inositol-3 kinase inhibitor, LY294002, previously demonstrated to be a pharmacological stimulus for sperm motility. Bicarbonate addition to spermatozoa results in a significant increase in sperm motility as well as in several hyperactivation parameters. This stimulatory effect of bicarbonate and LY294002 is mediated by an increase in cAMP production and tyrosine phosphorylation of the A kinase anchoring protein, AKAP3. The specificity of bicarbonate effects was confirmed by inhibition with 4,4′-di-isothiocyanostilbene-2,2′-disulfonic acid. We remark that, in human spermatozoa, bicarbonate acts primarily through activation of sAC to stimulate tyrosine phosphorylation of AKAP3 and sperm motility because both effects are blunted by the sAC inhibitor 2OH-estradiol. In conclusion, our data provide the first evidence that bicarbonate stimulates human sperm motility and hyperactivation through activation of sAC and tyrosine phosphorylation of AKAP3, finally leading to an increased recruitment of PKA to AKAP3.

Steroid feedback regulates GnRH secretion and previous work has implicated gamma-aminobutyric acid (GABA)ergic neurons as a mediator of these effects. We examined GABAergic postsynaptic currents (PSCs) in green fluorescent protein-identified GnRH neurons from mice exposed to different steroid milieus in vivo. Adult mice were ovariectomized and treated with estradiol (OVX E, controls) or E plus progesterone (P, OVX E P). P decreased PSC frequency, a presynaptic effect, and PSC size, which could be via pre- and/or postsynaptic mechanisms. In contrast, dihydrotestosterone (DHT, OVX E DHT) increased both GABAergic PSC frequency and size in GnRH neurons. Tetrodotoxin (TTX), which eliminates action-potential-dependent presynaptic effects, did not alter frequency, suggesting DHT may have increased PSC frequency by increasing connectivity between GABAergic and GnRH neurons. TTX reduced PSC size below control values, indicating DHT may augment presynaptic GABA release but inhibits the postsynaptic GnRH neuron response. In mice treated with both P and DHT (OVX E P DHT), PSC frequency and size were similar to controls, suggesting these steroids counteract one another. These results demonstrate GABAergic neurons participate in integrating and conveying steroid feedback to GnRH neurons, defining a potential central mechanism for steroid regulation of GnRH neurons during the reproductive cycle, and providing one possible mechanism for increased activity of these cells in hyperandrogenic females.

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.

PHTF1 is an 84–86-kDa membrane protein found in the endoplasmic reticulum of male germ cells in rodents. There are no evident signs of PHTF1 in the spermatozoa released into the lumen of the seminiferous tubules but PHTF1 is present in the epididymal epithelium. Characterization of the epididymal Phtf1 messenger by Northern blot and reverse transcription-PCR identified a 3-kilobase transcript in the epididymis, similar to that previously reported in the testis. The transcript is present in the proximal part of the epididymis and it appears when the rats reach 4 wk of age. Through immunofluorescence analysis, PHTF1 was localized in the principal cells of the initial segment and the caput epididymis. Colocalization with different markers indicated PHTF1 is in the endoplasmic reticulum saccules applied to the trans face of the Golgi system. Western blot analyses revealed a shorter form of the protein—about 56-kDa versus the 84-kDa form found in the testis. Using the canine epididymal cell line CIM 20, transfected by N- and C-terminal myc-tagged PHTF1, we demonstrated that the 56-kDa epididymal form could result from proteolytical processing.

The present study was designed to investigate the localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at various stages during the first meiosis were fixed and immunostained for MAD1, kinetochores, microtubules, and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes were treated with nocodazole or Taxol before examination. The anti-MAD1 antibody was injected into the oocytes at the germinal vesicle (GV) stage for examination of chromosome alignment and spindle formation. It was found that MAD1 was present in the oocytes from the GV to prometaphase I stages around the nuclei. When the oocytes reached the metaphase I (M-I) to metaphase II (M-II) stages, MAD1 was mainly localized at the spindle poles. However, MAD1 relocated to the vicinity of the chromosomes when spindles were disassembled by nocodazole or cooling, and the relocated MAD1 moved back to the spindle poles during spindle recovery. Taxol treatment did not affect the MAD1 localization. Although anti-MAD1 antibody injection did not affect nuclear maturation, significantly higher proportions of injected oocytes had misaligned chromosomes when the oocytes reached the M-I to M-II stages. The results of the present study indicate that MAD1 is present in mouse oocytes at all stages during the first meiosis and that it participates in spindle checkpoint during meiosis. However, MAD1 could not check misaligned chromosomes during spindle recovery after the spindles were destroyed by drug or cooling, which caused some chromosomes to scatter in the oocytes.

Endothelin (ET)-1 is a mitogenic factor in numerous cell types, including rat myometrial cells. In the present study, we investigated the potential role of ET-1 in the proliferation of tumoral uterine smooth muscle cells (ELT-3 cells). We found that ET-1 exerted a more potent mitogenic effect in ELT-3 cells than in normal myometrial cells, as indicated by the increase in [³H]thymidine incorporation, cell number, and bromodeoxyuridine incorporation. The ET-1 was more efficient than platelet-derived growth factor and epidermal growth factor to stimulate proliferation. The ET-1-mediated cell proliferation was inhibited in the presence of U0126, a specific inhibitor of (mitogen-activated protein kinase ERK kinase), indicating that extracellular signal-regulated kinase (ERK) activation is involved. Additionally, ET-1 induced the activation of phospholipase (PL) D, leading to the synthesis of phosphatidic acid (PA). The ET-1-induced activation of PLD was twofold higher in ELT-3 cells compared to that in normal cells. The two cell types expressed mRNA for PLD1a and PLD2, whereas PLD1b was expressed only in ELT-3 cells. The exposure of cells to butan-1-ol reduced ET-1-mediated production of PA by PLD and partially inhibited ERK activation and DNA synthesis. Addition of exogenous PLD or PA in the medium reproduced the effect of ET-1 on ERK activation and cell proliferation. Collectively, these data indicate that ET-1 is a potent mitogenic factor in ELT-3 cells via a signaling pathway involving a PLD-dependent activation of ERK. This highlights the potential role of ET-1 in the development of uterine leiomyoma, and it reinforces the role of PLD in tumor growth.

Sensitive and specific measurement of FSH is critical to research in reproductive biology, and the increasing availability of transgenic mouse models has created a need for a robust, sensitive, and specific mouse (m) FSH assay. The present study evaluated a time-resolved immunofluorometric assay (IFMA) for mFSH using monoclonal antibody to human (h) FSHβ as a capture antibody and a biotinylated polyclonal antibody to rat α subunit as a detection probe, with signaling amplified by europium-labeled streptavidin. The mFSH IFMA lowered the detection limit 34-fold (5 vs. 170 pg/sample) compared with standard mFSH RIA. The mFSH IFMA demonstrated parallelism of response to dilutions of castrated mouse serum and rat FSH but no cross-reactivity with hFSH and mLH or hLH, whereas the RIA demonstrated nonparallel cross-reactivity with hFSH. The IFMA has a wide analytical range, with a good precision profile for within- and between-assay reproducibility. Because the IFMA is a sandwich-type assay with strict dimer-specificity by design, the lower readings and recovery obtained were compared with the RIA when both assays used a pituitary-purified mFSH assay standard that contained isolated or fragmented subunits as well as intact dimeric FSH. When used with mouse serum sample, the mFSH IFMA demonstrated the expected increases following orchidectomy as well as markedly enhanced sensitivity to very low levels of endogenous mFSH in gonadotropin-deficient mice. Furthermore, the IFMA measured mFSH with fidelity in both intact and orchidectomized male mice without any interference from transgenic hFSH. The greatly enhanced sensitivity, specificity, and technical convenience of this mFSH IFMA will allow wider application of FSH measurements to very small blood samples in immature and mature mice as well as transgenic models.

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G₂ as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.

A relationship between extracellular Ca ², fowl sperm phospholipase A₂ activity, long-chain acylcarnitine content, and motility was demonstrated in previous work. Sperm motility appeared to depend upon Na -dependent Ca ² cycling when sperm were incubated at body temperature without glucose. In the present work, motility decreased as a function of time when sperm were incubated in 2 mM Ca ² prepared with either buffered isotonic sucrose or LiCl. However, this effect was less pronounced in the case of LiCl. The sparing effect of Li was attributed to the mitochondrial Na /Ca ² exchanger. Motile concentration decreased exponentially in response to micromolar concentrations of CGP 37157, a specific inhibitor of the mitochondrial Na /Ca ² exchanger. KB-R7943 mesylate, an inhibitor of the reverse mode of the Na /Ca ² exchanger, prevented re-initiation of motility when exogenous Ca ² was added to sperm rendered immotile by incubation with 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, a high-affinity Ca ² chelator. The presence of voltage-gated Ca ² channels was confirmed by the effect of nifedipine on motile concentration. Neither motile concentration nor straight line velocity was affected by either ouabain or orthovanadate, which inhibit Na -K ATPase and Ca ²-ATPase, respectively. In summary, we infer that 1) fowl sperm motility is dependent upon extracellular Ca ² cycling through mitochondria; 2) such cycling is dependent upon extracellular Na ; and 3) fowl sperm conserve ATP by moving neither Na nor Ca ² by active transport. Understanding the relationship between mitochondrial Ca ² cycling and ATP production may be applicable to long-term semen storage.

In the present study the authors investigated if the inhibitory effect of leptin in the ovary was mediated via nitric oxide (NO) using human granulosa cells (GCs). Human GCs were obtained from preovulatory follicles of women who underwent IVF. Reverse transcription–polymerase chain reaction (RT-PCR) demonstrated that human GCs expressed mRNA of leptin and mRNA of isoforms of leptin receptor, including one long form and two types of short forms. Exposure of human GCs to leptin at concentrations of 3–30 ng/ml for 60 min dose-dependently increased the fluorescence of 4,5-diaminofluorescein (DAF-2), an NO-sensitive dye. The effect of leptin on DAF-2 fluorescence was inhibited by pretreatment of human GCs with 100 μM nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase (NOS), indicating that the increase in DAF-2 fluorescence properly reflected the intracellular NO production. FSH (1 ng/ ml) and IGF-I (30 ng/ml) stimulated 17β-estradiol (E₂) production in human GCs, respectively. FSH plus IGF-I induced a further increase in E₂ production. Leptin did not significantly alter basal or FSH-dependent E₂ production, but it inhibited the effect of IGF-I on E₂ production and the synergistic effect of IGF-I on FSH-stimulated E₂ production. The inhibitory effect of leptin on IGF-I argumentation of E₂ production was attenuated by pretreatment of human GCs with 100 μM L-NAME. In conclusion, leptin could induce NO production in human GCs. The inhibitory effect of leptin on IGF-I augmentation of E₂ production in human GCs was attenuated by L-NAME, strongly suggesting that NO may mediate the action of leptin in human GCs.

Follicle-Stimulating Hormone (FSH) at a wide range of doses is routinely added to culture media during in vitro maturation (IVM) of oocytes, but the effects on oocyte health are unclear. The suggestion that superovulation may cause aneuploidy and fetal abnormalities prompted us to study the potential role of FSH in the genesis of chromosomal abnormalities during meiosis I. Mouse cumulus-oocyte complexes (COCs) isolated from the antral follicles of unprimed, sexually immature B6CBF₁ mice were cultured in increasing concentrations of FSH. Following culture, matured oocytes were isolated, spread, stained with DAPI, and the numbers of chromosomes counted. Significantly increased aneuploidy, arising during the first meiotic division, was observed in metaphase II oocytes matured in higher concentrations of FSH (≥20 ng/ml). The effect of FSH on spindle morphology and chromosome alignment during metaphase I was then explored using immunocytochemistry and three-dimensional reconstruction of confocal sections. High FSH had no effect on gross spindle morphology but did alter chromosome congression during prometaphase and metaphase, with the spread of chromosomes across the spindle at this time being significantly greater in oocytes cultured in 2000 ng/ml compared with 2 ng/ml FSH. Analysis of three-dimensional reconstructions of spindles in oocytes matured in 2000 ng/ml FSH shows that chromosomes are more scattered and farther apart than they are following maturation in 2 ng/ml FSH. These results demonstrate that exposure to high levels of FSH during IVM can accelerate nuclear maturation and induce chromosomal abnormalities and highlights the importance of the judicious use of FSH during IVM.

An inhibition of vitellogenesis is observed in fish exposed to cadmium (Cd), either in natural or in experimental conditions. To investigate whether this correlates or not with modifications in the expression of several genes involved in reproduction, we have performed a study on juvenile rainbow trout (Oncorhynchus mykiss) exposed to waterborne Cd in combination with estradiol (E₂). A relative reverse transcription-PCR protocol was used to evaluate the effect of Cd exposure on the expression of several genes. We quantified vitellogenin, rainbow trout estradiol receptor α (rtERα), short and long isoforms (rtERαS and rtERαL), mRNA levels in liver, and salmon GnRH1, salmon GnRH2, rtERαS, and rtERαL mRNA levels in the brain. In liver, Cd reduced the E₂-stimulated mRNA levels of vitellogenin as well as these of both rtERα isoforms in a dose-dependent manner. In brain tissue, our results indicate that rtERα mRNA levels are not enhanced by E₂. Cd treatments did not modify rtERαS isoform expression but reduced rtERαL expression in the brain. Focusing on the expression of salmon GnRH (sGnRH) genes, E₂ did not affect mRNA levels, but experiments with Cd alone greatly enhanced sGnRH 1 as well as sGnRH 2 gene expression in a dose-dependant manner. This study supports the idea that Cd is an important endocrine disrupter that could act through an inhibition of E₂-stimulated genes in the liver and also through a central effect on sGnRH gene expression. Cd may affect a number of E₂ signaling pathways but could also affect the reproductive axis by nonestrogenic mechanisms.

The present study was conducted to examine the effects of chondroitin sulfate A-derived oligosaccharide (ChSAO) on hyaluronidase activity and in vitro fertilization (IVF) parameters. The activity of hyaluronidase extracted from preincubated boar sperm was completely blocked by ChSAO at concentrations of 10 μg/ml or higher. After in vitro maturation of porcine cumulus-oocyte complexes, some oocytes were freed from their cumulus cells, and cumulus-intact or cumulus-free oocytes were inseminated with sperm in IVF medium containing various concentrations of ChSAO (0.1–100 μg/ml). In cumulus-intact oocytes, the penetration and the polyspermy rates (39% and 28%, respectively) were significantly decreased by treatment with 100 μg/ml ChSAO compared with those of oocytes treated without ChSAO (63% and 52%, respectively). On the contrary, in cumulus-free oocytes, the addition of 10–100 μg/ml ChSAO significantly reduced the polyspermy rate compared with the control (25–30% versus 53%, respectively), whereas ChSAO had no effect on sperm penetration. Interestingly, ChSAO added to IVF medium significantly decreased the number of sperm bound to the zona pellucida (ZP) of cumulus-free oocytes in a concentration-dependent manner between 0.1 and 100 μg/ml. However, ChSAO had no effect on the time course change in ZP modification after oocyte activation by electrostimulation and the incidence of the acrosome-reacted sperm. Treatment with 100 μg/ml ChSAO during IVF of cumulus-free oocytes significantly increased the proportion of development to the blastocyst stage after in vitro insemination. Therefore, the present findings indicate that hyaluronidase-inhibitory ChSAO is an efficient probe for promoting normal fertilization process in terms of an effective decrease in the incidence of polyspermy during IVF of porcine oocytes.

We previously reported the discovery of a novel mammalian H1 linker histone termed H1FOO (formerly H1OO), a replacement H1, the expression of which is restricted to the growing/ maturing oocyte and to the zygote. The significance of this pre-embryonic H1 draws on its substantial orthologous conservation, singular structural attributes, selectivity for the germ cell lineage, prolonged nucleosomal residence, and apparent predominance among germ cell H1s. Herein, we report that the intronic, single-copy, five-exon (≥5301 base pair) H1foo gene maps to chromosome 6 and that the corresponding primary H1foo transcript gives rise to two distinct, alternatively spliced mRNA species (H1foo_α and H1foo_β). The expression of the oocytic H1FOO transcript and protein proved temporally coupled to the recruitment of resting primordial follicles into a developing primary follicular cohort and thus to the critical transition marking the onset of oocytic growth. The corresponding potential protein isoforms (H1FOO_α and H1FOO_β), both nuclear localization sequence-endowed but export consensus sequence-free and possessing a significant net positive charge, localized primarily to perinucleolar heterochromatin in the oocytic germinal vesicle. Further investigation will be required to define the functional role of the H1FOO protein in the ordering of the chromatin of early mammalian development as well as its potential role in defining the primordial-to-primary follicle transition.

Our aim was to study the inhibitory and facilitatory factors possibly accounting for the undetectable activity of the GnRH pulse generator in late fetal life in vitro and its awakening in early postnatal life. Gamma aminobutyric acid (GABA_A) receptor antagonism using SR 95 531 did not cause any secretory pulse in fetal explants, whereas a significant stimulation of GnRH pulse frequency was obtained at 5 and 15 days. GnRH secretory response to repeated N-methyl-d-aspartate (NMDA) stimulation showed progressive disappearance, indicating that the inhibitory autofeedback was operating. GnRH release caused by glutamine was respectively 9% and 20% of that evoked by glutamate in fetal and 5-day-old rats whereas both amino acids were equally active at 15 days. Explants obtained after cesarean section performed at onset of labor did not show any secretory pulse, while pulses could be observed with explants obtained 2 h after vaginal delivery. Incubation of fetal explants with oxytocin (10⁻⁸ M) or prostaglandin E₂ (PGE₂) (10⁻⁶ M) resulted in occurrence of GnRH secretory pulses. A facilitatory effect of the oxytocin was shown to persist on Days 1, 5, and 15 and inhibitory effects of an oxytocin receptor antagonist provided some evidence of endogenous oxytocin involvement. We conclude that, in the fetal rat hypothalamus, GnRH inhibitory autofeedback and GABAergic inputs do not account for the absence of pulsatile GnRH secretion in vitro. A low rate of glutamate biosynthesis from glutamine is a possibly limiting factor. Oxytocin and PGE₂ can play a facilitatory role in the postpartal occurrence of pulsatile GnRH secretion.

Whether endocrine disruption in an individual male is actually translated into reduced reproductive success in a natural competitive environment is extremely difficult to predict. Here, we have used paternity analysis to provide new information on the ability of an endocrine disruptor to deleteriously affect male guppy reproductive fitness by including the effect of intermale competition. Groups of male guppies were exposed to 10.5, 44.4, or 112 ng/L of the synthetic estrogen 17α-ethinylestradiol (EE2) from birth to adulthood. Subsequently, an exposed male competed against an unexposed male for the opportunity to fertilize a receptive female. The successful males siring the majority of the offspring in each brood were then identified using microsatellites in genetic paternity analysis. Only the highest dose of EE2 produced harmful effects with a significantly female-biased sex ratio, significant reductions in male sperm count, testis weight, body coloration and courtship behavior, and a significant increase in body size. These feminizing effects were translated into a highly significant reduction in fertility, where only 1 of the 17 exposed males sired offspring in competition with unexposed males. The evidence suggests that EE2-treated males have reduced reproductive fitness compared with untreated males, possibly the result of EE2 effects on multiple fitness traits. To our knowledge, this is the first study providing evidence of endocrine disruption at the population level that has included the ecologically highly relevant effect of sexual competition on male reproductive fitness.

Acrylamide is an animal carcinogen and probable human carcinogen present in appreciable amounts in heated carbohydrate-rich foodstuffs. It is also a germ cell mutagen, inducing dominant lethal mutations and heritable chromosomal translocations in postmeiotic sperm of treated mice. Acrylamide's affinity for male germ cells has sometimes been overlooked in assessing its toxicity and defining human health risks. Previous investigations of acrylamide's germ cell activity in mice showed stronger effects after repeated administration of low doses compared with a single high dose, suggesting the possible involvement of a stable metabolite. A key oxidative metabolite of acrylamide is the epoxide glycidamide, generated by cytochrome P4502E1 (CYP2E1). To explore the role of CYP2E1 metabolism in the germ cell mutagenicity of acrylamide, CYP2E1-null and wild-type male mice were treated by intraperitoneal injection with 0, 12.5, 25, or 50 mg acrylamide (5 ml saline)⁻¹ kg⁻¹ day⁻¹ for 5 consecutive days. At defined times after exposure, males were mated to untreated B6C3F₁ females. Females were killed in late gestation and uterine contents were examined. Dose-related increases in resorption moles (chromosomally aberrant embryos) and decreases in the numbers of pregnant females and the proportion of living fetuses were seen in females mated to acrylamide-treated wild-type mice. No changes in any fertility parameters were seen in females mated to acrylamide-treated CYP2E1-null mice. Our results constitute the first unequivocal demonstration that acrylamide-induced germ cell mutations in male mice require CYP2E1-mediated epoxidation of acrylamide. Thus, CYP2E1 polymorphisms in human populations, resulting in variable enzyme metabolic activities, may produce differential susceptibilities to acrylamide toxicities.

Sperm capacitation is a maturation process, occurring in the female reproductive tract, that produces fertilization-competent spermatozoa. Protein tyrosine phosphorylation represents an important event in capacitation. The present study demonstrates the capacitation-dependent tyrosine-phosphorylation of phospholipid hydroperoxide glutathione peroxidase (PHGPx), the disulfide cross-linked, major structural protein of the sperm mitochondrial capsule. Immunofluorescence microscopy using an antiphosphotyrosine monoclonal antibody (anti-pY20) demonstrated the presence of capacitation-associated tyrosine phosphorylated proteins in the flagellum of hamster spermatozoa. Among the tyrosine-phosphorylated polypeptides (M_r 19 000– 99 000), a 19-kDa polypeptide was the only one that can be solubilized completely by Triton X-100-dithiothreitol (DTT). The 19-kDa polypeptide was purified by anion-exchange chromatography and by immunoaffinity chromatography. Proteomic identification of the 19-kDa polypeptide by nano-electrospray tandem mass spectrometry yielded six peptides that matched the National Center for Biotechnology Information (NCBI) database sequences of bovine PHGPx. Indirect immunofluorescence localized PHGPx to the midpiece of the flagellum and the immunoblot analysis demonstrated its DTT-dependent release from purified flagella. DTT extracts of noncapacitated spermatozoa exhibited a charge train of four major PHGPx isoforms (pIs 7.5– 9.0) by two-dimensional PAGE, whereas capacitated spermatozoa revealed the generation of new acidic PHGPx isoforms with isoelectric points ranging between pH 6.0–7.0 and 4.0–5.0, indicating that it is posttranslationally modified during capacitation. These data suggest that the tyrosine-phosphorylation of PHGPx may represent an important event in the signaling pathway(s) associated with capacitation and could potentially affect mitochondrial function.

Betaglycan was originally characterized as the type III receptor for TGFβ, yet recent research has indicated that betaglycan can serve as an accessory receptor for inhibin. To understand better the action of inhibin in avian follicular development, we have investigated the expression of betaglycan in the pituitary gland and ovary of the hen. In experiments 1 and 2, betaglycan mRNA was detected at 6 kilobases (kb) by Northern blot analysis (n = 5) in chicken pituitary, granulosa, and theca layers and whole ovary. Expression of betaglycan was greatest in the pituitary gland in experiment 1 and greater in the granulosa layer of small yellow follicles (SYF) compared with the granulosa layer of larger follicles. In experiment 2, betaglycan mRNA was more abundantly expressed in the theca layer compared with the granulosa layer for all follicle sizes, although there was no significant difference in betaglycan expression in the theca layer among follicle sizes. In experiment 3, immunohistochemical analysis revealed betaglycan protein in the anterior pituitary as well as in the ovary (n = 4) and SYF (n = 4). Colocalization studies revealed a high abundance of cells within the anterior pituitary expressing both betaglycan and FSH (n = 4). Betaglycan protein was found in the granulosa layer; however, markedly enhanced staining was observed in the theca layer of ovarian follicles. Our results provide evidence for expression of betaglycan mRNA and protein colocalization with FSH in the anterior pituitary, consistent with known inhibin effects. Ovarian localization of betaglycan, particularly in the theca layer, suggests a paracrine role for inhibin in the hen.

One-cell-stage embryos derived from most random-bred and inbred female mice exhibit an in vitro developmental block at the two-cell stage in classical embryo culture media. However, embryos derived from many F₁ hybrids develop easily past the two-cell stage under the same conditions. This has given rise to the commonly accepted idea that there exist blocking and nonblocking types of female mice, with only the former being prone to a two-cell block. Recently, culture media have been improved to the point that even embryos prone to the two-cell block will develop past the block in vitro, making it possible to study its etiology. Here, we show that either increased osmolarity or increased glucose/phosphate levels induced the expected two-cell block in random-bred CF1 embryos and the two-cell block at increased osmolarities could be rescued by the organic osmolyte glycine. Surprisingly, one-cell embryos from B6D2F₁ (BDF₁) F₁ hybrid females, considered to be nonblocking, also became blocked at the two-cell stage when osmolarity or glucose/phosphate levels were increased. They were also similarly rescued by glycine from the osmolarity-induced block. The most evident difference was that the purportedly nonblocking embryos became blocked at a higher threshold of osmolarity or glucose/phosphate level than those considered prone to this developmental block. Thus, both blocking and nonblocking embryos actually exhibit a similar two-cell block to development.

A master regulator of DNA replication, CDC6 also functions in the DNA-replication checkpoint by preventing DNA rereplication. Cyclin-dependent kinases (CDKs) regulate the amount and localization of CDC6 throughout the cell cycle; CDC6 phosphorylation after DNA replication initiation leads to its proteolysis in yeast or translocation to the cytoplasm in mammals. Overexpression of CDC6 during the late S phase prevents entry into the M phase by activating CHEK1 kinase that then inactivates CDK1/cyclin B, which is essential for the G₂/M-phase transition. We analyzed the role of CDC6 during resumption of meiosis in mouse oocytes, which are arrested in the first meiotic prophase with low CDK1/cyclin B activity; this is similar to somatic cells at the G₂/M-phase border. Overexpression of CDC6 in mouse oocytes does not prevent resumption of meiosis. The RNA interference-mediated knockdown of CDC6, however, reveals a new and unexpected function for CDC6; namely, it is essential for spindle formation in mouse oocytes.

In mouse ovaries, growth differentiation factor-9 (GDF9) is an oocyte-derived growth factor that plays an essential role during early follicular development. However, the role of GDF9 during later stages of follicular development is uncertain. In the present study, a long double-stranded (ds) RNA interference approach was used to investigate the possible role of GDF9 in mediating oocyte regulation of cumulus expansion. Fully grown mouse oocytes injected with Gdf9 dsRNA, Bmp15 dsRNA, or injection buffer were cultured for 24 h and processed for measurement of Gdf9 and Bmp15 mRNA levels using real-time reverse transcription-polymerase chain reaction (RT-PCR) and for measurement of GDF9 protein levels using Western blot analysis and immunofluorescence. Injection with Gdf9 dsRNA knocked down Gdf9, but not Bmp15, mRNA expression in oocytes, and vice versa. Furthermore, GDF9 protein levels were reduced in the Gdf9 dsRNA-injected oocytes. To investigate the role of GDF9 in cumulus expansion, two endpoints were used to evaluate cumulus expansion: Has2 and Ptgs2 mRNA levels were measured in cumulus cells using real-time RT-PCR, and assessment of cumulus expansion was undertaken morphologically. After 24 h of culture in the presence of 0.5 IU/ml of FSH, cumulus shells cocultured with buffer- and Bmp15 dsRNA-injected oocytes exhibited a high degree of expansion, whereas cumulus shells cocultured with Gdf9 dsRNA-injected oocytes exhibited only limited expansion. Supporting this observation, Has2 and Ptgs2 mRNA levels after 8 h of coculture were lower in cumulus cells cocultured with Gdf9 dsRNA-injected oocytes than in those cocultured with buffer-injected oocytes. The present results strongly support the concept that GDF9 is a key mediator of oocyte-enabled cumulus expansion in mice.

A number of substances have been implicated in the regulation of oxytocin (OT) secretion from bovine corpus luteum in vivo. However, isolated bovine luteal cells cultured in a monolayer lose the ability to secrete OT in response to stimulatory substances. The present study investigated how cell-to-cell contact and the cytoskeleton affect OT secretion by isolated bovine luteal cells. In experiment 1, bovine midluteal cells (Days 8–12 of the estrous cycle) were stimulated with prostaglandin F_2α (PGF_2α; 1 μM), noradrenaline (NA; 10 μM), or growth hormone (GH; 5 nM) in two culture systems: In one system, cell monolayers were incubated in 24-well culture plates, and in the other system, aggregates of cells were incubated in glass tubes in a shaking water bath. The cells cultured in a monolayer underwent considerable spreading and showed a variety of shapes, whereas the cells cultured in glass tubes remained fully rounded during the experimental period and soon formed aggregates of cells. Although PGF_2α, NA, and GH did not stimulate OT secretion by the monolayer cells, all tested substances stimulated OT secretion by the aggregated cells (P < 0.01). In experiment 2, the monolayer cells were pre-exposed for 1 h to an antimicrofilament agent (cytochalasin B; 1 μM) or two antimicrotubule agents (colchicine or vinblastine; 1 μM) before stimulation with PGF_2α, NA, or GH. Although PGF_2α, NA, and GH did not stimulate OT secretion by the monolayer cells in the presence of colchicine or vinblastine, they all stimulated OT secretion in the presence of cytochalasin B (P < 0.001). The overall results show that OT secretion by bovine luteal cells depends on microfilament function and cell shape. Moreover, the aggregate culture system that allows three-dimensional, cell-to-cell contact seems to be a good model for studying OT secretion by isolated bovine luteal cells.

The consequence of mono-(2-ethylhexyl) phthalate (MEHP)-induced injury of testicular Sertoli cells is the Fas-dependent apoptotic elimination of germ cells. In addition to the well-known ability of p53 to regulate the transcription of various apoptosis-associated proteins, p53 also has been implicated in mediating the localization of Fas to the plasma membrane of various cell types in a transcription-independent manner. To resolve the role of p53 in MEHP-mediated testicular toxicity, we used wild-type (p53 ^/ ) and p53 knockout (p53^−/−) mice. A significantly lower incidence of TUNEL-positive germ cells was observed in p53^−/− mice compared to p53 ^/ mice at 1, 1.5, and 24 h after MEHP exposure. In these same mice, an induction of Fas and death receptor-5 (DR5) in testicular membrane preparations was observed only in p53 ^/ mice. Analyses of mRNA levels in testes of p53 ^/ and p53^−/− mice by reverse transcription-polymerase chain reaction revealed that increases in membrane levels of Fas occurred in the absence of their transcriptional up-regulation. Processing of procaspase-8 was observed only in MEHP-treated p53 ^/ mice, and this correlated with the observed incidence of germ cell apoptosis. Interestingly, the p53 status of mice also influenced the stability of c-FLIP (L), a caspase-8 inhibitory protein, that was measured at levels approximately two- to fivefold higher in p53^−/− mice after MEHP-exposure compared to those in p53 ^/ mice. Taken together, these data suggest that MEHP-induced germ cell apoptosis is dependent, in part, on the p53 protein and on its abilities to increase the localization of Fas and DR5 on the germ cell membrane as well as to decrease the cellular levels of c-FLIP (L).

The field of genomics applies the dissection of genetic differences toward an understanding of the biology of complex traits. Quantitative trait loci (QTL) for testis size, plasma FSH in boars, and body composition (backfat) have been identified near the centromere on the X chromosome in a Meishan–White Composite resource population. Since thyroid function affects Sertoli cell development and adult testis size in rodents, and thyroxine-binding globulin (TBG) maps to this region on the porcine X chromosome, TBG was a positional candidate gene for testis size. We discovered a polymorphism in exon 2 of the porcine TBG gene that results in an amino acid change of the consensus histidine to an asparagine. This single nucleotide polymorphism (SNP) resides in the ligand-binding domain of the mature polypeptide, and the Meishan allele is the conserved allele found in human, bovine, sheep, and rodent TBG. Binding studies indicate altered binding characteristics of the allelic variants of TBG with the asparagine (White Composite) isoform having significantly greater affinity for thyroxine than the histidine (Meishan) isoform. Alternate alleles in boars from the resource population are also significantly associated with testis weight. Therefore, this polymorphism in TBG is a candidate for the causative variation affecting testis size in boars.

Prenatal exposure of the female sheep to excess testosterone (T) leads to hypergonadotropism, multifollicular ovaries, and progressive loss of reproductive cycles. We have determined that prenatal T treatment delays the latency of the estradiol (E₂)-induced LH surge. To extend this finding into a natural physiological context, the present study was conducted to determine if the malprogrammed surge mechanism alters the reproductive cycle. Specifically, we wished to determine if prenatal T treatment 1) delays the onset of the preovulatory gonadotropin surge during the natural follicular phase rise in E₂, 2) alters pulsatile LH secretion and the dynamics of the secondary FSH surge, and 3) compromises the ensuing luteal function. Females prenatally T-treated from Day 60 to Day 90 of gestation (147 days is term) and control females were studied when they were ∼2.5 yr of age. Reproductive cycles of control and prenatally T-treated females were synchronized with PGF_2α, and peripheral blood samples were collected every 2 h for 120 h to characterize cyclic changes in E₂, LH, and FSH and then daily for 14 days to monitor changes in luteal progesterone. To assess LH pulse patterns, blood samples were also collected frequently (each 5 min for 6 h) during the follicular and luteal phases of the cycle. The results revealed that, in prenatally T-treated females, 1) the preovulatory increase in E₂ was normal; 2) the latencies between the preovulatory increase in E₂ and the peaks of the primary LH and FSH surges were longer, but the magnitudes similar; 3) follicular-phase LH pulse frequency was increased; 4) the interval between the primary and secondary FSH surges was reduced but there was a tendency for an increase in duration of the secondary FSH surge; but 5) luteal progesterone patterns were in general unaltered. Thus, exposure of the female to excess T before birth produces perturbances and maltiming in periovulatory gonadotropin secretory dynamics, but these do not produce apparent defects in cycle regularity or luteal function. To reveal the pathologies that lead to the eventual subfertility arising from excess T exposure during midgestation, studies at older ages must be conducted to assess if there is progressive disruption of neuroendocrine and ovarian function.

Prelabor rupture of the fetal membranes affects approximately 10% of women at term, resulting in an increased risk of maternal and neonatal infection. Evidence suggests that membrane rupture is related to biochemical processes involving the extracellular matrix of the membranes. We tested the hypothesis that prelabor ruptured membranes are characterized by reduced collagen concentrations, altered collagen cross-link profiles, and increased concentrations of biomarkers of oxidative damage. We also set out to determine whether these effects are modulated by ascorbic acid status. In a case-control study, we explored the role that ascorbic acid, oxidative stress, collagen, and collagen cross-links play in determining membrane integrity and developed a functional assay to assess membrane proteolytic susceptibility. Prelabor ruptured membrane had a reduced ascorbic acid concentration in comparison with controls while protein carbonyl and malondialdehyde concentrations were increased. Collagen concentrations were also reduced in prelabor ruptured membrane, and while the concentration of collagen cross-links was not significantly different between prelabor and timely ruptured membrane, there was a regional variation in cross-link ratio within the amniotic sac. Proteolytic resistance in vitro was reduced in prelabor ruptured membrane and also exhibited regional variation within the amniotic sac. Our findings are strongly supportive of a role for the enhanced degradation of membrane collagen in the determination of prelabor rupture of fetal membranes. The formation of the rupture initiation site is a function of a regional variation in collagen cross-link ratio. Tissue ascorbic acid status may be an important mediator of these processes.

Spermatogenesis originates from a small population of spermatogonial stem cells. These cells are believed to divide infinitely and support spermatogenesis throughout life in the male. In this investigation, we examined the possibility of deriving transgenic offspring from single spermatogonial stem cells. Spermatogonial stem cells were transfected in vitro with a plasmid vector containing a drug resistant gene. Stably transfected stem cell clones were isolated by in vitro drug selection; these clones were expanded and used to produce transgenic progeny following spermatogonial transplantation into infertile recipients. An average of 49% of the offspring carried the transgene, and the recipient mice continued to produce monoclonal transgenic progeny a year after transplantation. Thus, a somatic cell-based genetic approach can be used to modify and select clones of spermatogonial stem cells in a manner similar to embryonic stem cells. The feasibility of genetic selection using postnatal spermatogonial stem cells demonstrates their extensive proliferative potential and provides the opportunity to develop new methods for generating stable animal transgenics or for germline gene therapy.

The assembly of the developmentally arrested primordial follicle and subsequent transition to the primary follicle are poorly understood processes critical to ovarian biology. Abnormal primordial follicle development can lead to pathologies such as premature ovarian failure. The current study used a genome-wide expression profile to investigate primordial follicle assembly and development. Rat ovaries with predominantly unassembled, primordial, or primary follicles were obtained. RNA from these ovaries was hybridized to rat microarray gene chips, and the gene expression (i.e., ovarian transcriptome) was compared between the developmental stages. Analysis of the ovarian transcriptome demonstrated 148 genes up-regulated and 50 genes down-regulated between the unassembled and primordial follicle stages. Observations demonstrate 80 genes up-regulated and 44 genes down-regulated between the primordial and primary follicle stages. The analysis demonstrated 2332 genes common among the three developmental stages, 146 genes specific for the unassembled follicles, 94 genes specific for the primordial follicles, and 151 genes specific for the primary follicles. Steroidogenic genes are up-regulated between unassembled and primordial follicles, and then many are again down-regulated between primordial and primary follicles. The hormones inhibin and Müllerian inhibitory substance (MIS) display a similar pattern of expression with the highest levels of mRNA in the primordial follicles. Several novel unknown genes that had dramatic changes in expression during primordial follicle development were also identified. Gene families/clusters identified that were up-regulated from unassembled to primordial follicles include growth factors and signal transduction gene clusters, whereas a down-regulated gene family was the synaptonemal complex genes associated with meiosis. Gene families/clusters that were up-regulated between primordial and primary follicles included immune response genes, metabolic enzymes, and proteases, whereas down-regulated gene families include the globulin genes and some steroidogenic genes. The expression of several growth factors changed during primordial follicle development, including vascular endothelial growth factor and insulin-like growth factor II. Elucidation of how these changes in gene expression coordinate primordial follicle assembly and the primordial to primary follicle transition provides a better understanding of these critical biological processes and allows selection of candidate regulatory factors for further investigation.