Maternal uteroplacental blood flow increases during pregnancy. Altered uteroplacental blood flow is a core predictor of abnormal pregnancy. Normally, the uteroplacental arteries are invaded by endovascular trophoblast and remodeled into dilated, inelastic tubes without maternal vasomotor control. Disturbed remodeling is associated with maintenance of high uteroplacental vascular resistance and intrauterine growth restriction (IUGR) and preeclampsia. Herein, we review routes, mechanisms, and control of endovascular trophoblast invasion. The reviewed data suggest that endovascular trophoblast invasion involves a side route of interstitial invasion. Failure of vascular invasion is preceded by impaired interstitial trophoblast invasion. Extravillous trophoblast synthesis of nitric oxide is discussed in relation to arterial dilation that paves the way for endovascular trophoblast. Moreover, molecular mimicry of invading trophoblast-expressing endothelial adhesion molecules is discussed in relation to replacement of endothelium by trophoblast. Also, maternal uterine endothelial cells actively prepare endovascular invasion by expression of selectins that enable trophoblast to adhere to maternal endothelium. Finally, the mother can prevent endovascular invasion by activated macrophage-induced apoptosis of trophoblast. These data are partially controversial because of methodological restrictions associated with limitations of human tissue investigations and animal studies. Animal models require special care when extrapolating data to the human due to extreme species variations regarding trophoblast invasion. Basal plates of delivered placentas or curettage specimens have been used to describe failure of trophoblast invasion associated with IUGR and preeclampsia; however, they are unsuitable for these kinds of studies, since they do not include the area of pathogenic events, i.e., the placental bed.

Hedgehog (Hh) proteins are expressed during vertebrate development in some tissues with inductive properties and at epithelial-mesenchymal boundaries in several developing organs, including the lung, gut, hair follicle, and tooth. The Hh signaling pathway is highly conserved, and important clues to understanding the mechanism of Hh signal transduction in vertebrates have come from studies in Drosophila. In recent years, Hh signaling has been recognized during embryonic development and in some cases during postnatal life in several mammalian tissues whose functions are essential for reproduction, including the gonads, uterus, and hormonally responsive accessory sex glands such as the prostate and mammary gland. The role of the pathway in these tissues is highly reminiscent of its role at epithelial-mesenchymal-stromal boundaries in other organ systems, which has provided a framework within which to explore Hh signaling in tissues that function in reproduction. Some features unique to these tissues are emerging, including a role in proliferation and differentiation of male germline cells in mammals and apparent influences of sex steroids on Hh signaling. However, many questions remain about the function of Hh signaling in the gonads, uterus, prostate, and mammary gland, including factors regulating the signal transduction pathway, identification of downstream target genes, and roles for Hh signaling in diseases involving these tissues.

Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase catalytic reverse transcriptase subunit (hTERT) in these cells reconstitutes telomerase activity and immortalizes the cells without tumor transformation. In this report, we show that sheep fibroblasts are similar to human cells. They do not have detectable telomerase activity and undergo only a finite numbers of cell divisions before replicative senescence. Telomere lengths in sheep fibroblasts are similar to those reported for human cells and shorten at a rate of 50–200 base pairs (bp) each cell division. Expression of the human telomerase catalytic subunit restored the telomerase activity in the sheep cells and extended their proliferative life span. None of the telomerase positive sheep fibroblasts exhibited a transformed phenotype after 200 days of continuous culture, and the higher hTERT expressing cells maintained their telomere lengths and normal cell characteristics for more than 500 days in culture. In cloning experiments using one of these cell lines as a nuclear donor, the reconstructed karyoplasts were reprogrammed and developed to the blastocyst stage at a similar frequency to that observed with the parental, telomerase negative cell line. After embryo transfer the blastocysts exhibited a relatively high frequency of implantation, early fetal development, and organogenesis. No fetuses survived beyond 40 days of development, however, showing that although these cells could be substantially reprogrammed, they were not fully competent for nuclear transfer.

It has been suggested that locally produced insulin-like growth factor binding protein 4 (IGFBP4) inhibits ovarian follicular growth and ovulation by interfering with IGF action. According to this hypothesis, IGFBP4-expressing follicles should demonstrate atresia, whereas healthy dominant follicles should be devoid of IGFBP4. Alternatively, according to this view, there could be constitutive expression of the inhibitory IGFBP4 but selective expression of an IGFBP4 protease in dominant follicles, allowing the follicle to mature and ovulate because of degradation of the binding protein. To examine these views concerning the role of IGFBP4 in primate follicular selection, we analyzed cellular patterns of IGFs 1 and 2, IGFBP4, and the IGFBP4 protease (pregnancy-associated plasma protein A [PAPP-A]) mRNA expression in ovaries from late follicular phase rhesus monkeys using in situ hybridization. The IGF1 mRNA was not detected, but the IGF2 mRNA was abundant in theca interna and externa of all antral follicles and was present in the granulosa of large preovulatory and ovulatory follicles. The IGFBP4 mRNA was selectively expressed by LH receptor (LHR) mRNA-positive theca interna cells of healthy antral follicles (defined by aromatase and gonadotropin receptor expression) and by LHR-expressing granulosa cells found only in large preovulatory and ovulatory follicles (defined by size and aromatase expression). The PAPP-A mRNA was abundant in granulosa cells of most follicles without obvious relation to IGFBP4 expression. Ovarian IGFBP4 mRNA levels were markedly increased after treatment with the LH analog, hCG, whereas IGF2 and PAPP-A mRNAs were not significantly altered. In summary, IGFBP4 expression appears to be associated with follicular selection, not with atresia, in the monkey ovary. The IGFBP4 is consistently expressed in healthy theca interna and in luteinized granulosa cells, likely under LH regulation. The IGFBP4 protease, PAPP-A, is widely expressed without apparent selectivity for IGFBP4-expressing follicles or for dominant follicles. These observations suggest that IGFBP4 or an IGFBP4 proteolytic product may be involved with LH-induced steroidogenesis and/or luteinization rather than with inhibition of follicular growth.

Seasonal fluctuations in immune status have been documented for avian and mammalian populations. During the late summer and early fall, immune function is bolstered to help animals cope with the more physiologically demanding winter. The environmental cue for these seasonal changes is apparently decreasing photoperiod. In the present study, we determined the potential role of leptin in mediating the effect of photoperiod on cell-mediated immune responses in male mice. Leptin-deficient (ob/ob) and littermate control mice were housed for 10 wk in either a short (8L:16D) or a long (16L:8D) photoperiod beginning at 6 wk of age. After the mice were killed, immune and reproductive organs were weighed and splenocytes isolated. The proliferative and cytokine responses (interleukin [IL]-2 and IL-4) of splenocytes to the T-cell mitogen, concanavalin A (Con A; 0–40 μg/ml), were determined. Body weights were elevated and both testes and seminal vesicle weights subnormal in ob/ob mice (by ANOVA, main effect of leptin deficiency), but thymuses and spleens were of normal size. Serum leptin levels were at minimum detection limits in ob/ob mice, but leptin levels in control mice housed at 8L:16D were higher than in control mice housed at 16L:8D. The proliferative response of splenocytes from ob/ob mice to Con A was subnormal (by ANOVA, main effect of leptin deficiency), but photoperiod had no effect on this response. Production of IL-2 in splenocytes of ob/ob mice was subnormal (by ANOVA, main effect of leptin deficiency) irrespective of photoperiod, but cells from mice housed at 8L:16D (by ANOVA, main effect of photoperiod) produced more IL-2 than cells from animals housed at 16L:8D. In contrast, a leptin deficiency did not alter IL-4 production, but cells from animals (ob/ob and controls) housed at 16L:8D produced less IL-4 than cells from animals housed at 8L:16D (by ANOVA, main effect of photoperiod). The present study suggests that both photoperiod and leptin have mutually independent effects on the proliferation of lymphocytes and cytokine production profiles. The data do not provide definitive support for the hypothesis that photoperiod-induced changes in leptin secretion mediate the effects of season on immune status.

During spermatogenesis, diploid stem cells differentiate, undergo meiosis and spermiogenesis, and transform into haploid spermatozoa. Various factors have been demonstrated to regulate this marvelous process of differentiation, but the expression of only a few genes specifically involved in spermatogenesis has been studied. In the present study, different types of spermatogenic cells were isolated from Balb/c mice testes of different ages using the velocity sedimentation method, and we determined the expression profiles of 1176 known mouse genes in six different types of mouse spermatogenic cells (primitive type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, round spermatids, and elongating spermatids) using Atlas cDNA arrays. Of the 1176 genes on the Atlas Mouse 1.2 cDNA Expression Arrays, we detected 181 genes in primitive type A spermatogonia, 256 in type B spermatogonia, 221 in preleptotene spermatocytes, 160 in pachytene spermatocytes, 141 in round spermatids, and 126 in elongating spermatids. A number of genes were detected as differential expression (up-regulation or down-regulation). Fourteen of the differentially expressed genes have been further confirmed by reverse transcription-polymerase chain reaction for their expression characterizations in different types of spermatogenic cells. These results provide more information for further studies into spermatogenesis-related genes and may lead to the identification of genes with potential relevance to spermatogenesis.

Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence the donor cell program could lead to altered embryonic phenotypes. Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos. Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos. Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface. Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake. These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development. As a result, cloned embryos initially exhibit many somatic cell-like characteristics. Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference. We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.

Ovarian changes determined by daily transrectal ultrasound and its relationship with FSH, LH, estradiol-17β, progesterone, and inhibin were investigated in six goats for three consecutive interovulatory intervals. Estrous cycles were synchronized using two injections of prostaglandin F_2α analogue 11 days apart. All follicles 3 mm or greater in diameter and corpora lutea were measured daily. A follicular wave was defined as one or more follicles growing to 5 mm or greater in diameter. The day that the follicles reached 3 mm in diameter was defined as the day of wave emergence, and the first wave after ovulation was defined as wave 1. During the interovulatory interval (mean ± SEM, 21.3 ± 0.4 days; n = 18), follicular waves emerged at 0.3 ± 0.5, 6.5 ± 0.2, and 12.1 ± 0.4 days for wave 1, wave 2, and wave 3, respectively, in goats with three waves of follicular development and at −0.6 ± 0.3, 4.7 ± 0.2, 9.4 ± 0.5, and 13.4 ± 0.5 days for wave 1, wave 2, wave 3, and wave 4, respectively, in goats with four waves of follicular development (Day 0 = the day of ovulation). The mean diameter of the largest follicle of the ovulatory wave was significantly larger than those of the largest follicles of the other waves. Corpora lutea could be identified ultrasonically at Day 3 postovulation and attained 12.1 ± 0.3 mm in diameter on Day 8. Transient increases in plasma concentrations of FSH were detected around the day of follicular wave emergence. The level of FSH was negatively correlated with that of inhibin. These results demonstrated that follicular waves occurred in goats and that the predominant follicular wave pattern was four waves with ovulation from wave 4. These results also suggested that the emergence of follicular waves was closely associated with increased secretion of FSH.

The activator protein-1 (AP-1) transcription factors are important regulators of cell proliferation and differentiation. The developmental distribution of AP-1 family members in porcine ovary has not been previously investigated. We examined the expression of AP-1 factors in porcine ovarian follicles, granulosa cells, and corpora lutea at different stages of development. Immunoblot analyses confirmed that c-Jun, JunD, JunB, c-Fos, Fra-1, Fra-2, and FosB immunoreactive proteins were present in whole-cell extracts (WCE) of all antral follicles and midluteal phase corpora lutea (CL) as well as granulosa cells (GC) isolated from different-sized antral follicles. The intensities of c-Jun and c-Fos protein bands were decreased in CL WCE compared to antral follicles. In granulosa cells from preovulatory follicles (8–10 mm), Fra-2 exhibited a shift from 43 kDa to 46 kDa when compared to granulosa cells from smaller antral follicles. Separation of cytoplasmic and nuclear extracts was performed to determine if developmental differences between these fractions existed. Most AP-1 factors predominated in the nuclear fraction with notable exceptions. c-Fos predominated in the nucleus in GC and follicles but predominated in the cytoplasmic fraction of CL. With the exception of GC from 1–2-mm follicles, in which expression was similar between fractions, Fos-B was found predominantly in the cytoplasmic fraction. Fra-1 exhibited similar expression between cytoplasmic and nuclear fractions for all tissues. Immunohistochemical (IHC) analyses of porcine ovary sections were performed to determine the cellular distribution of these factors at different follicular stages, and immunopositive nuclei were evaluated. In primordial and primary unilaminar follicles, all AP-1 factors studied except for FosB were detected in granulosa nuclei. Granulosa cell nuclei of multilaminar preantral follicles were immunopositive for all factors, with lower expression of FosB. Antral follicles exhibited GC and thecal cell nuclear staining for all factors with the exception of FosB in theca. Luteal cells exhibited the most intense nuclear staining for JunD and Fra-2, whereas all other factors were present in luteal cell nuclei although to a lesser extent. IHC with FosB antibodies yielded mostly cytoplasmic staining but only weak luteal nuclear staining. In corpora albicantia, low levels of staining were seen for all AP-1 factors. The DNA-binding abilities of these factors in granulosa cells and CL were evaluated by EMSA. Nuclear extracts from granulosa cells from 1–2-mm or 8–10-mm antral follicles bound an AP-1 DNA consensus sequence and complexes consisted predominantly of c-Jun, JunD, JunB, c-Fos, and Fra-2. In CL, c-Jun, JunD, JunB, and Fra-2 were present in DNA-binding complexes, and c-Fos binding was not detected. In conclusion, our results suggest that expression and DNA-binding activity of AP-1 factors in follicular structures changes with luteinization. Differentiation to the luteal phenotype involves a reduction in nuclear c-Jun and c-Fos and a predominance of JunD and Fra-2.

The luteinizing hormone receptor (LHR) plays an essential role as a mediator of LH and CG action during embryonic sexual differentiation and in gametogenesis. In a hypogonadal male patient, we recently demonstrated that a genomic deletion of exon 10, located in the hinge region of the extracellular domain, results in discrimination of LH and hCG action. In the common marmoset (Calltithrix jacchus), exon 10 of the LHR is naturally missing at the mRNA level. In order to investigate whether this is an isolated species-specific phenomenon, we performed a phylogenetic screening, searching for the presence of LHR exon 10 mRNA in a number of primate species representative for the major lineages of primate evolution. The expressed LHR region encompassing exon 10 was amplified from testicular tissue by RT-PCR, cloned, and sequenced. In addition, we performed Southern blot analysis of the LHR of selected New World and Old World primates. The results revealed that exon 10 mRNA is lacking in the complete New World monkey (Platyrrhini) lineage but is present in both more primitive and more advanced primates. However, exon 10 seems to be present at the genomic level, arguing for a splicing failure possibly due to a genomic mutation or the lack of appropriate splicing factors. Considering that, in the human, LH is far less active than hCG on the LHR lacking exon 10, we addressed the question whether the existence of such a receptor has any consequences on the dual hormone LH/CG system present in Platyrrhini. Using primers specific for the known marmoset CG β cDNA, we amplified the CG β subunit cDNA from male common marmoset pituitaries by RT-PCR, while LH β could not be amplified, suggesting a possible physiological role of pituitary CG in this species. In conclusion, we demonstrated for the first time that the LH mRNA without exon10 is the natural wild-type LHR in the Platyrrhini lineage. We propose that this LHR represents a new subclass of receptors that should be named LHR type II. In addition, the high expression of CG β in the marmoset pituitary suggests a physiological role of CG in the reproductive function of these primates beyond pregnancy.

The central circadian clock in mammals is housed in the brain and is based on cyclic transcription and translation of clock proteins. How the central clock regulates peripheral organ function is unclear. However, cyclic expression of circadian genes in peripheral tissues is well established, suggesting that these tissues have their own endogenous oscillators. Reproduction is a process influenced by circadian rhythms in many organisms, thus making the testis an attractive model for studying clock function in peripheral organs. However, results addressing cyclic expression of clock genes in the mammalian testis are inconsistent. To resolve this issue, RNA was extracted from testes of mice at various times of day. Expression of the circadian clock genes mPer1, mPer2, Bmal1, Clock, mCry1, and Npas2 was constant at all times. Immunohistochemical localization of mPER1 and CLOCK proteins revealed restricted expression only in cells at specific developmental stages of spermatogenesis. For mPER1, these stages are the spermatogonia and the condensing spermatids. In contrast, CLOCK expression was restricted to round spermatids, specifically within the developing acrosome. Expression of mPER1 and CLOCK was constant at all times of day. These results suggest that clock proteins have noncircadian functions in spermatogenesis. Noncircadian expression of clock genes was also found in the thymus, which, like the testis, is composed primarily of differentiating cells. We propose that cyclic expression of clock genes is suspended during cellular differentiation.

Osteopontin (OPN) is a phosphorylated and glycosylated, secreted protein that is present in various epithelial cells and biological fluids. On freezing and thawing or treatment with proteases, the native 70-kDa protein gives rise to 45- and 24-kDa fragments. Secreted OPN functions as an extracellular matrix (ECM) protein that binds cell surface receptors to mediate cell-cell adhesion, cell-ECM communication, and cell migration. In sheep and humans, OPN is proposed to be a secretory product of uterine glandular epithelium (GE) that binds to uterine luminal epithelium (LE) and conceptus trophectoderm to mediate conceptus attachment, which is essential to maintain pregnancy through the peri-implantation period. Cell-cell adhesion, communication, and migration likely are important at the interface between uterus and placenta throughout pregnancy, but to our knowledge, endometrial and/or placental expression of OPN beyond the peri-implantation period has not been documented in sheep. Therefore, the present study determined temporal and spatial alterations in OPN mRNA and protein expression in the ovine uterus between Days 25 and 120 of pregnancy. The OPN mRNA in total ovine endometrium increased 30-fold between Days 40 and 80 of gestation. In situ hybridization and immunofluorescence analyses revealed that the predominant source of OPN mRNA and protein throughout pregnancy was the uterine GE. Interestingly, the 45-kDa form of OPN was detected exclusively, continuously, and abundantly along the apical surface of LE, on conceptus trophectoderm, and along the uterine-placental interface of both interplacentomal and placentomal regions through Day 120 of pregnancy. The 45-kDa OPN is a proteolytic cleavage fragment of the native 70-kDa OPN, and it is the most abundant form in uterine flushes during early pregnancy. The 45-kDa OPN is more stimulatory to cell attachment and cell migration than the native 70-kDa protein. Collectively, the present results support the hypothesis that ovine OPN is a component of histotroph secreted by the uterine GE that accumulates at the uterine-placental interface to influence maternal-fetal interactions throughout gestation in sheep.

Ovulation (i.e., the release of mature oocytes from the ovary) requires spatially targeted follicle rupture at the apex. Both progesterone and prostaglandins play key roles in the ovulatory process. We have studied follicle rupture and ovulation in adult cycling rats treated with a progesterone receptor antagonist (RU486), an inhibitor of prostaglandin synthesis (indomethacin, IM), or both. All rats were treated with LHRH antagonist on the morning (0900 h) of proestrus to inhibit endogenous gonadotropins and with 10 μg of ovine LH (oLH) at 1700 h in proestrus to induce ovulation. Animals were treated from metestrus to proestrus with 2 mg/day of RU486 or vehicle (olive oil) and on the morning of proestrus (1200 h) with 1 mg of IM or vehicle (olive oil). Some rats treated with vehicle or RU486 were killed on the morning of proestrus to assess preovulatory follicle development. The remaining rats were killed on the morning of estrus to study follicle rupture and ovulation. In vehicle-treated rats, oLH induced ovulation in 98% of follicles. In IM-treated rats, spatial targeting of follicle rupture was disrupted. Most oocytes were released to the ovarian interstitium (50%) or to the periovarian space (39%), and a smaller percentage (11%) of oocytes remained trapped inside the luteinized follicle. RU486-treated rats showed, on the morning of estrus, unruptured luteinized follicles. Only occasionally (2.8%), the oocytes were released to the periovarian space. IM treatment induced follicle rupture in RU486-treated rats, and 25% of oocytes were released to the ovarian interstitium. However, the number of oocytes released to the periovarian space (i.e., ovulated) was not increased by IM treatment in rats lacking progesterone actions. Overall, these data indicate that RU486 and IM have opposite effects on follicle rupture and suggest that both progesterone and prostaglandins are necessary for the spatial targeting of follicle rupture at the apex.

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca² ]_i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca² transients, but inhibitors of P/Q type (ω-agatoxin; ω-conotoxin MVIIC), N-type (ω-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the α_1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (±) BAY K 8644 induced [Ca² ]_i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca² ]_i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca² with Mn² allowed Mn² influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.

In mammalian cells, essential polyunsaturated fatty acids (PUFAs) are converted to longer PUFAs by alternating steps of elongation and desaturation. In contrast to other PUFA-rich tissues, the testis is continuously drained of these fatty acids as spermatozoa are transported to the epididymis. Alteration of the germ cell lipid profile from spermatogonia to condensing spermatids and mature spermatozoa has been described, but the male gonadal gene expression of the desaturases, responsible for the PUFA-metabolism, is still not established. The focus of this study was to characterize the expression and regulation of stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), and Δ⁵- and Δ⁶-desaturase in rat testis. Desaturase gene expression was detected in testis, epididymis, and separated cells from seminiferous tubulus using Northern blot analysis. For the first time, SCD1 and SCD2 expression is demonstrated in rat testis and epididymis, both SCDs are expressed in epididymis, while testis mainly contains SCD2. Examination of the testicular distribution of Δ⁵- and Δ⁶-desaturase and SCD1 and SCD2 shows that all four desaturases seem to be localized in the Sertoli cells, with far lower expression in germ cells. In light of earlier published results showing that germ cells are richer in PUFAs than Sertoli cells, this strengthens the hypothesis of a lipid transport from the Sertoli cells to the germ cells. As opposed to what is shown in liver, Δ⁵- and Δ⁶-desaturase mRNA levels in Sertoli cells are up-regulated by dexamethasone. Furthermore, dexamethasone induces SCD2 mRNA. Insulin also up-regulates these three genes in the Sertoli cell, while SCD1 mRNA is down-regulated by both insulin and dexamethasone. Δ⁵- and Δ⁶-desaturase, SCD1, and SCD2 are all up-regulated by FSH. A similar up-regulation of the desaturases is observed when treating Sertoli cells with (Bu)₂cAMP, indicating that the desaturase up-regulation observed with FSH treatment results from elevated levels of cAMP. Finally, testosterone has no influence on the desaturase gene expression. Thus, FSH seems to be a key regulator of the desaturase expression in the Sertoli cell.

During the period when they are producing sperm, male sea lampreys (Petromyzon marinus L.) release a sex pheromone 7α, 12α, 24-trihydroxy-5α-cholan-3-one-24-sulfate (3 keto-petromyzonol sulfate, 3ketoPZS) that induces search and preference behaviors in ovulating females. In this study, we conducted a series of experiments to demonstrate that release of this pheromone into water takes place exclusively through the gills. In a behavioral maze, water conditioned with the anterior region of spermiating males induced an increase of search and preference behaviors in ovulating females. Similar behavior was not elicited by water conditioned by the posterior region. The anterior region washings and whole-body washings from spermiating males also elicited large and virtually identical electro-olfactogram responses from female sea lampreys, while the posterior washings produced negligible responses. Further, mass spectrometry and immunoassay confirmed that virtually all the 3ketoPZS released into water was through the gills. Immunocytochemistry revealed some gill epithelial cells and hepatocytes from spermiating males contained dense immunoreactive 3ketoPZS, but not those from prespermiating males. These results demonstrate that 3ketoPZS is released through the gill epithelia and suggest that this pheromone or its precursor may be produced in the liver.

Early gestation is critical for placentomal growth, differentiation, and vascularization, as well as fetal organogenesis. The fetal origins of adult disease hypothesis proposes that alterations in fetal nutrition and endocrine status result in developmental adaptations that permanently change structure, physiology, and metabolism, thereby predisposing individuals to cardiovascular, metabolic, and endocrine disease in adult life. Multiparous ewes were fed to 50% (nutrient restricted) or 100% (control fed) of total digestible nutrients from Days 28 to 78 of gestation. All ewes were weighed weekly and diets adjusted for individual weight loss or gain. Ewes were killed on Day 78 of gestation and gravid uteri recovered. Fetal body and organ weights were determined, and numbers, morphologies, diameters, and weights of all placentomes were obtained. From Day 28 to Day 78, restricted ewes lost 7.4% of body weight, while control ewes gained 7.5%. Maternal and fetal blood glucose concentrations were reduced in restricted versus control pregnancies. Fetuses were markedly smaller in the restricted group than in the control group. Further, restricted fetuses exhibited greater right- and left-ventricular and liver weights per unit fetal weight than control fetuses. No treatment differences were observed in any gross placentomal measurement. However, caruncular vascularity was enhanced in conceptuses from nutrient-restricted ewes but only in twin pregnancies. While these alterations in fetal/placental development may be beneficial to early fetal survival in the face of a nutrient restriction, their effects later in gestation as well as in postnatal life need further investigation.

This study tested the hypothesis that endocrinological threshold levels of progesterone that induce negative feedback effects on the pulsatile and surge modes of LH secretion are different. Our approach was to examine the effects of subnormal progesterone concentrations on LH secretion. Long-term ovariectomized Shiba goats that had received implants of silastic capsules containing estradiol were divided into three groups. The high progesterone (high P) group received a subcutaneous implant of a silastic packet (50 × 70 mm) containing progesterone, and the low progesterone (low P) group received a similar implant of a small packet (25 × 40 mm) containing progesterone. The control (non-P) group received no treatment with exogenous progesterone. Blood samples were collected daily throughout the experiment for the analysis of gonadal steroid hormone levels and at 10-min intervals for 8 h on Days 0, 3, and 7 (Day 0: just before progesterone treatment) for analysis of the pulsatile frequency of LH secretion. Then estradiol was infused into the jugular vein of all animals at a rate of 3 μg/h for 16 h on Day 8 to determine whether an LH surge was induced. Blood samples were collected every 2 h from 4 h before the start of the estradiol infusion until 48 h after the start of the infusion. In each group, the mean ± SEM concentration after progesterone implant treatment was 3.3 ± 0.1 ng/ml for the high P group, 1.1 ± 0.1 ng/ml for the low P group, and <0.1 ng/ml for the non-P group, concentrations similar to the luteal levels, subluteal levels, and follicular phase levels of the normal estrous cycle, respectively. The estradiol concentration ranged from 4 to 8 pg/ml after estradiol capsule implants in all groups. The LH pulse frequency was significantly (P < 0.05) suppressed on Day 3 (6.2 ± 0.5 pulses/8 h) and on Day 7 (2.6 ± 0.9 pulses/8 h) relative to Day 0 (9.0 ± 0.5 pulses/8 h) in the high P group. In both the low P and non-P groups, however, the changes of pulsatile frequency of LH were not significantly different, and high pulses (7–9 pulses/8 h) were maintained on each of the 3 days they were tested. An LH surge (peak concentration, 100.3 ± 11.0 ng/ml) occurred in all goats in the non-P group, whereas there was no surge mode secretion of LH in either the high P or the low P group. The results of this study support our hypothesis that the threshold levels of progesterone that regulate negative feedback action on the LH pulse and the LH surge are different. Low levels of progesterone, around 1 ng/ml, completely suppressed the LH surge but did not affect the pulsatile frequency of LH secretion.

Cloning by somatic cell nuclear transfer is inefficient. This is evident in the significant attrition in the number of surviving cloned offspring at virtually all stages of embryonic and fetal development. We find that cloned preimplantation mouse embryos aberrantly express the somatic form of the Dnmt1 DNA (cytosine-5) methyltransferase, the expression of which is normally prevented by a posttranscriptional mechanism. Additionally, the maternal oocyte-derived Dnmt1o isoform undergoes little or none of its expected translocation to embryonic nuclei at the eight-cell stage. Such defects in the regulation of Dnmt1s and Dnmt1o expression and cytoplasmic-nuclear trafficking may prevent clones from completing essential early developmental events. Furthermore, aberrant Dnmt1 localization and expression may contribute to the defects in DNA methylation and the developmental abnormalities seen in cloned mammals.

Dax1 is an orphan nuclear receptor expressed in both Leydig and Sertoli cells of the testis. Mutation of DAX1 in humans causes adrenal failure and hypogonadotropic hypogonadism. Targeted mutagenesis of Dax1 in mice reveals a primary gonadal defect characterized by overexpression of aromatase and cellular obstruction of the seminiferous tubules and efferent ductules, leading to germ cell death and infertility. Transgenic expression of DAX1 under the control of the müllerian-inhibiting substance promoter, which is selectively expressed in Sertoli cells, improves fertility but does not fully correct the histological abnormalities in the testes of Dax1 knockout (Dax1KO) mice. We therefore hypothesized that Dax1 may also play a crucial role in other somatic cells of the testis, namely the Leydig cells. A 2.1-kilobase fragment of the murine LH receptor 5′-promoter (LHR-DAX1) was used to generate transgenic mice that selectively express DAX1 in Leydig cells. Expression of the LHR-DAX1 transgene caused no observable phenotype in wild-type mice but improved fertility when expressed in Dax1KO males (rescue [RS]). Although testicular size was not increased in LHR-DAX1 RS animals, aromatase expression was restored to normal levels, and sperm production was increased. Testicular pathology was only slightly improved in RS mice compared to Dax1KO animals. Taken together with the result of previous studies of DAX1 expression in Sertoli cells, we conclude that the testis phenotype of Dax1KO mice reflects the combined effects of Dax1 deficiency in both Sertoli and Leydig cells.

The germ cell-deficient (gcd) mouse mutation is a recessive, transgenic insertional mutation associated with the disruption of two Chr11 genes, Pog (proliferation of germ cells) and Vrk2 (vaccinia virus-related protein kinase 2). We have recently shown that like gcd/gcd mice, targeted Pog^−/− males and females show virtually no spermatogenesis or oogenesis at 4–6 wk of age. Because Pog is deleted in gcd/gcd and Pog^−/− mice, a comparison of the phenotypes of the two mouse models is appropriate. Here, we report that unlike in POG-deficient females, the germ cells in POG-deficient males eventually populate the seminiferous tubules at 9 wk, and fertility can be achieved by 12 wk. Homozygous gcd/gcd males did not show a similar degree of germ cell population, and most gcd/gcd males remained infertile at 16 and 22 wk of age. A comparison of the degree of germ cell deficiency at 13.5 days postcoitum and 1 day postpartum between Pog^−/− and gcd/gcd males revealed that gcd/gcd males had far fewer germ cells than Pog^−/− males at both time points. Our data suggest that Pog is essential for proper primordial germ cell proliferation in the embryonic stage but is not needed for spermatogonial proliferation after birth. Thus, the difference in the spermatogenetic potential in adult Pog^−/− and gcd/gcd mice may result from the severity of germ cell deficiency rather than from the inability of gcd/gcd spermatogonia to proliferate efficiently. The greater deficiency of germ cells before the onset of spermatogenesis seen in gcd/gcd males compared to Pog^−/− mice suggests either that the different background affects the outcome of Pog deletion or that Vrk2 has additional effects on germ cell development.

It has been shown that mature oocytes injected with nuclei from round spermatids collected from mouse testis can generate normal offspring and that round spermatids can develop in vitro. An undetermined issue is whether spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (γ-H2AX), a marker for DNA double-strand breaks. Cocultured round spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round spermatids developed in vitro were documented by videomicroscopy ( http://www.sci.ccny.cuny.edu/∼kier). We also show that breeding of male and female mouse offspring generated by spermatid nuclear injection produced fertile offspring. In addition to their capacity to produce fertile offspring, cocultured, flagellated round spermatids can facilitate the analysis of the mechanisms of centriolar polarity, duplication, assembly, and flagellar growth, including the intraflagellar transport of cargo proteins.

After ejaculation, mammalian spermatozoa must undergo capacitation to fertilize. Capacitation of bovine spermatozoa occurs in vitro in medium supplemented with heparin. Semen cryopreservation is an important tool for assisted reproduction, although the fertility of frozen-thawed spermatozoa is reduced, possibly due to precocious capacitation-like changes that are known to occur. Our purpose was to clarify the mechanisms involved in bull sperm cryocapacitation induced by cryopreservation. Our general hypothesis is that the signaling pathways that lead to capacitation are triggered by the cryopreservation procedure. Ejaculated bovine semen was divided into two aliquots and diluted in extender; one was then kept fresh, whereas the second was cryopreserved. Western blots of extracted sperm proteins with anti-phosphotyrosine antibody showed that capacitation, induced by either heparin in fresh sperm or cryopreservation (cryocapacitation), is associated with a differential profile of phosphotyrosine-containing proteins. Immunolocalization of phosphotyrosine-containing proteins in the fresh and cryopreserved spermatozoa showed that, after thawing, cryocapacitated sperm displayed labeling over the acrosomal region, whereas for fresh sperm, this labeling appeared after 5-h incubation with heparin. The chlortetracycline assay and the ability of the sperm to undergo the lysophosphatidylcholine-induced acrosome reaction were used to confirm that a subpopulation of cryopreserved sperm is capacitated at thawing, irrespective of heparin inclusion. Since glucose is known to inhibit heparin-induced capacitation, the semen extender was modified to include glucose as a means of inhibiting cryocapacitation; however, cryocapacitation was not prevented according to the chlortetracycline assay and profile of phosphotyrosine-containing sperm proteins.

Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and examined by confocal laser scanning microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle and the first polar body. After sperm injection, an aster of microtubules formed adjacent to the sperm head and subsequently enlarged such that at the time of pronucleus migration and apposition it filled the entire cytoplasm. During syngamy, the microtubule matrix reorganized to form a mitotic spindle on which the chromatin of both parents aligned. Finally, after nuclear and cellular cleavage were complete, the microtubule asters dispersed into the interphase daughter cells. Sham injection induced parthenogenetic activation of 76% of oocytes, marked by the formation of multiple cytoplasmic microtubular foci that later developed into a dense microtubule network surrounding the female pronucleus. The finding that a parthenote alone can produce a microtubule aster, whereas the aster invariably forms at the base of the sperm head during normal fertilization, indicates that both gametes contribute to the formation of the zygotic centrosome in the horse. Finally, 25% of sperm-injected oocytes failed to complete fertilization, mostly due to absence of oocyte activation (65%), which was often accompanied by failure of sperm decondensation. In conclusion, this study demonstrated that union of the parental genomes in horse zygotes is accompanied by a series of integrated cytoskeleton-mediated events, failure of which results in developmental arrest.

To determine the prostaglandin (PG) H₂ synthase (generally referred to as cyclooxygenase [COX]) isozyme responsible for producing uterotonic PGs during parturition, we used PGF_2α receptor-deficient mice, which exhibit parturition failure due to impaired withdrawal of serum progesterone at term. On ovariectomy-induced parturition in these mice, uterine COX-2 mRNA expression was drastically induced in the myometrium, whereas COX-1 mRNA expression in the endometrial epithelium decreased. The concomitant administration of progesterone with ovariectomy resulted in a delay in parturition and the disappearance of both the increase in COX-2 mRNA and the decrease in COX-1 mRNA. Thus, the expression of myometrial COX-2 and the occurrence of parturition are closely associated in this model. Furthermore, administration of the COX-nonselective inhibitor, indomethacin, or the COX-2-selective inhibitor, Dup-697 or JTE-522, effectively delayed ovariectomy-induced parturition in these mice. These findings suggest that COX-2-derived PGs contribute to the onset of parturition after the decrease in serum progesterone level.

Ischemia-reperfusion (IR) of the testis results in germ cell-specific apoptosis and can lead to aspermatogenesis. Germ cell-specific apoptosis after IR of the testis has been shown to be correlated with and dependent on neutrophil recruitment to the testis after IR. Studies that used E-selectin-deficient mice have demonstrated that E-selectin expression is critical for neutrophil recruitment to subtunical venules in the testis after IR and for the resultant germ cell-specific apoptosis. The present study investigates the in vivo signaling pathway that exists after IR that leads to neutrophil recruitment in the murine testis. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. Results demonstrate that the proinflammatory cytokines, tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β), are stimulated after IR as is the phosphorylation of c-jun N-terminal kinase (JNK). The downstream transcription factors of JNK, ATF-2 and c-jun are also phosphorylated at specific times after IR of the testis. Activation of the JNK stress-related kinase pathway is correlated with an increase in E-selectin expression and neutrophil recruitment to the testis after IR. Intratesticular injection of IL-1β also caused JNK phosphorylation and neutrophil recruitment to the testis. These results suggest that testicular IR injury stimulates IL-1β expression, which leads to activation of the JNK signaling pathway and ultimately E-selectin expression and neutrophil recruitment to the testis. This provides the first evidence of a cytokine/stress-related kinase signaling pathway to E-selectin expression in vivo.

Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine 1 (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.

Two experiments were conducted to examine circulating concentrations of progesterone (P₄) in cows with ovarian follicular cysts (OFCs) and to relate differing levels of P₄ to subsequent follicular events. In experiment 1, peripheral concentrations of P₄ were determined in cows diagnosed with OFCs. Nonpregnant, lactating Holstein and Jersey cows (n = 32) were diagnosed as having OFCs by rectal palpation. Ovarian follicular cysts were then examined by transrectal ultrasonography to confirm the presence of OFCs (follicle diameter, ≥17 mm; absence of luteal tissue). At confirmation, a blood sample was collected for quantification of P₄. The concentration of P₄ at confirmation was classified as low (<0.1 ng/ml), intermediate (0.1–1.0 ng/ml), or high (1.0–2.0 ng/ml). More OFCs were associated with intermediate (66%) than with either low (28%) or high (6%) concentrations of P₄. In experiment 2, the fate of follicles (diameter, ≥10 mm) that formed in the presence of an OFC was determined and related to circulating concentrations of P₄ during follicular development. Follicles (n = 59) that formed in the presence of an OFC ovulated (n = 19), formed a cyst (n = 30), or underwent normal growth and regression (NGR; n = 10). Endogenous P₄ in the 7-day period during follicular development was classified as low (if P₄ dropped to <0.1 ng/ml for 1 day or longer), intermediate (if P₄ averaged between 0.1 and 1.0 ng/ml and never dropped to <0.1 ng/ml), or high (if P₄ averaged >1.0 ng/ml and never dropped to <0.1 ng/ml). In the presence of intermediate P₄, 75% of observed follicles formed cysts, compared with 10% that ovulated and 15% that experienced NGR. In the presence of low P₄, 53%, 41%, and 6% of follicles ovulated, formed a follicular cyst, or experienced NGR, respectively. Thus, an association between intermediate P₄ and the formation of OFCs was established.

Eosinophils are prevalent in the female reproductive tract, where they may contribute to regulation of development and maintenance of epithelial integrity. The present study examined the effects of constitutive interleukin-5 (IL-5) expression and overabundance of eosinophils on the development and function of the mammary gland, uterus, and ovary in mice. Eosinophils were up to 13-fold and 4-fold more abundant in the uterus and mammary gland, respectively, in female IL-5 transgenic (IL-5Tg) mice than in wild-type (Wt) animals. Eosinophils were present in large numbers in regressing corpora lutea in IL-5Tg mice but not in ovaries from Wt mice. Postpubertal mammary gland development was retarded in IL-5Tg mice, with impaired terminal end bud formation and an altered pattern of epithelial cell proliferation across the mammary fat pad coincident with disrupted ductal branching and extension. By 10 wk of age, the ductal tree was complete in both genotypes. Onset of first estrus was also delayed in IL-5Tg mice, but once IL-5Tg mice reached puberty, serum estrogen content across the cycle and estrous cycle duration were normal. The histology of uterine tissue and epithelial cell turnover were unchanged. Capacity to mate and achieve pregnancy was not affected by maternal IL-5 transgene expression, although at Day 18 of gestation, a modest decrease in the fetal:placental weight ratio was observed. Furthermore, parturition and ability to lactate and nurture postnatal pup development were not compromised. These data demonstrate an effect of IL-5 overexpression on ductal morphogenesis during postpubertal mammary gland development that is consistent with a direct regulatory role for eosinophils in these events, but these data also show that eosinophil excess does not have long-term consequences for adult reproductive function.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to differentially regulate the expression of the gonadotropin subunit genes in cultures of rat pituitary cells. PACAP is expressed within the hypothalamus, and concentrations of PACAP are 2- to 4-fold higher in the portal circulation than in the general circulation. Therefore, PACAP is a candidate regulator of pituitary function. In the present study, we examined the expression of PACAP mRNA within the paraventricular nucleus (PVN) during maturation (Days 20–60) in the male rat and compared this expression to the levels of the gonadotropin subunits, follistatin, and GnRH-receptor mRNAs within the anterior pituitary. Serum concentrations of FSH and LH confirm the established maturational pattern of divergent secretion of LH and FSH. Northern analysis of the gonadotropin subunit mRNAs revealed that FSHβ expression parallels FSH secretion whereas LHβ mRNA concentrations do not change during development. Expression of the GnRH receptor in the pituitary parallels that of FSHβ. In situ hybridization revealed a developmental pattern of PACAP mRNA within the PVN that is reciprocal to that of FSHβ. Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total pituitary follistatin mRNA revealed no significant changes; however, semiquantitative RT-PCR analyses revealed the presence of two follistatin mRNA species, one of which, corresponding to follistatin-288, was developmentally regulated. These studies identified a reciprocal relationship between PVN PACAP and FSHβ gene expression in maturing rats. We propose that PACAP contributes to the selective regulation of FSHβ expression during maturation in the male rat, perhaps via regulation of follistatin.

The toxic effects of i.v. administration of N-acetyl-l-cysteine (NAC), a component of parenteral nutrition solutions, on fertility and embryonic development were investigated in SD male and female rats at doses of 100, 300, and 1000 mg kg⁻¹ day⁻¹. Infertility was observed in females in the 1000-mg/kg group throughout the period from before mating to embryogenesis. No effect of NAC on the reproductive ability of the male rats was seen. The oocytes and embryos were assessed morphologically to clarify the cause of the effects of NAC. The unfertilized oocytes (UO) recovered from the ampullae of the uterine tubes and Gestational Day (GD) 1 and 2 embryos recovered from the oviducts or uterus of the rats that received NAC i.v. at a dosage of 1000 mg kg⁻¹ day⁻¹ for more than 1 wk before mating were assessed morphologically by stereomicroscopy. In addition, the thickness of the zona pellucida (ZP) was calculated by morphometric evaluation of the UO. Fewer UO were collected in the NAC group than in the control (nontreatment) group. Interestingly, ZP-lacking or partially ZP-lacking oocytes were observed in the NAC group, and the morphometric evaluation of the UO showed thinning of the ZP. The number of embryos in each animal was markedly decreased on GD1, and no embryos were recovered on GD2 in the NAC group. The oocytes that had ZP affected by NAC treatment were abnormal or nonviable. The findings of the present study suggest that changes in the ZP are related to the infertility associated with NAC.

The mechanism of sperm storage in the fowl oviduct has remained a mystery since the 1960s, when sperm storage tubules (SST) were discovered between the shell gland and vagina. Previously, it was known that only motile sperm could ascend the vagina and enter these tubules. However, the means by which sperm resided therein was not clear. Research with computer-assisted sperm motion analysis has demonstrated that 1) seminal plasma glutamate acts as a motility agonist via N-methyl-d-aspartate receptors; 2) motility depends on extracellular Ca² and Na ; 3) straight-line velocity is a variable with a skewed distribution; 4) sperm cell trajectory is a function of straight-line velocity; and 5) specific inhibition of phospholipase A₂ renders sperm immotile. An additional experiment demonstrated that Ca² acts as a second messenger and thereby modulates the content of long-chain acylcarnitine within sperm. Therefore, it is proposed that 1) the release of endogenous fatty acids fuels sperm as they ascend the vagina; (2) on entering the SST, motile sperm maintain position against a fluid current generated by SST epithelial cells; 3) resident sperm metabolize exogenous fatty acids released from lipid-laden epithelial cells; (4) motile sperm emerge from the SST when their velocity declines to a threshold at which retrograde movement begins; and 5) the skewed distribution of straight-line velocity accounts for the exponential pattern of sperm emergence from the SST. In summary, sperm residence within and emergence from the SST are phenomena most likely explicable in terms of sperm cell motility.

Ubiquitination of the sperm mitochondria during spermatogenesis has been implicated in the targeted degradation of paternal mitochondria after fertilization, a mechanism proposed to promote the predominantly maternal inheritance of mitochondrial DNA in humans and animals. The identity of ubiquitinated substrates in the sperm mitochondria is not known. In the present study, we show that prohibitin, a highly conserved, 30- to 32-kDa mitochondrial membrane protein, occurs in a number of unexpected isoforms, ranging from 64 to greater than 185 kDa in the mammalian sperm mitochondria, which are the ubiquitinated substrates. These bands bind antiubiquitin antibodies, displaying a pattern consistent with polyubiquitinated “ladders.” Immunoprecipitation of sperm extracts with antiprohibitin antibodies followed by probing of the resultant immunocomplexes with antiubiquitin yields a banding pattern identical to that observed by antiprohibitin Western blot analysis. In fact, the presumably nonubiquitinated 30-kDa prohibitin band shows no antiubiquitin immunoreactivity. We demonstrate that ubiquitination of prohibitin occurs in testicular spermatids and spermatozoa. Ubiquitinated prohibitin molecules also accumulate in the defective fractions of ejaculated spermatozoa, which are thought to undergo surface ubiquitination during epididymal passage. In such sperm fractions, ubiquitin also coprecipitates with tubulin and microtubule-associated proteins, presumably contributed by the axonemes of defective, ubiquitinated spermatozoa. The results of the present study suggest that prohibitin is one of the ubiquitinated substrates that makes the sperm mitochondria recognizable by the egg's ubiquitin-proteasome dependent proteolytic machinery after fertilization and most likely facilitates the marking of defective spermatozoa in the epididymis for degradation.

Translational control of specific mRNAs by cytoplasmic polyadenylation has fundamental roles in gametogenesis. The cytoplasmic polyadenylation element binding (CPEB) protein regulates cytoplasmic polyadenylation of mRNAs as a trans factor in oogenesis and spermatogenesis. The CPEB protein contains two RNA recognition motifs and a Zn-finger structure. Proteins (KIAA0940 and KIAA1673) with similar structures are predicted from the genome database, but nothing is known about their expression and function. Here, we report another novel member of the CPEB protein family, CPEB2. Comparison of the amino acid sequences of CPEB family members suggests that the family can be divided structurally and, perhaps, functionally into two groups: the CPEB group, and the CPEB2-KIAA0940-KIAA1673 group. The CPEB2 maps to mouse chromosome distal 5B and is abundantly expressed in testis. However, it was detected by reverse transcription-polymerase chain reaction in all tissues that we examined. It preferentially binds to poly(U) and localizes to the cytoplasm in transfected HeLa cells. The CPEB2 is expressed postmeiotically in mouse spermatogenesis, suggesting a possible role in translational regulation of stored mRNAs in transcriptionally inactive haploid spermatids.

In response to the gonadotropin surge, the compact cumulus-oocyte complex (COC) undergoes expansion by synthesis of the mucopolysaccharide hyaluronan (HA) accompanying oocyte maturation. The objective of the present study was to quantify mRNA transcripts of the HA synthase (HAS) 1, HAS2, and HAS3 and the HA-receptors CD44 and RHAMM (receptor for HA-mediated motility). Additionally, we determined the histological localization of HA and its receptor, CD44, in maturing bovine COCs and cultured granulosa cells (GCs). Full-length transcript of bovine HAS2 and a part of the bovine RHAMM sequence has been made available. Real-time reverse transcriptase-polymerase chain reaction was used for individual mRNA expressions of bovine COCs in comparison to follicular GC gonadotropin treatment. Localization of CD44 and HA were done by immunohistochemistry and biotinylated HA-binding protein, respectively. Gonadotropins caused a rapid, 120-fold increase of HAS2 mRNA, whereas a delayed, 2-fold up-regulation of HAS3 mRNA was observed. The HAS1 transcripts were barely detected. Expression of CD44 mRNA greatly increased during in vitro maturation of COCs, indicating an important role when compared to an unchanged, steady-state RHAMM expression. As a consequence, HA was locally enriched after COC expansion, but only limited change was observed in the GCs. In cultured GCs, HAS2 expression was stimulated through FSH application, followed by the effective treatments of FSH LH and LH. Treatment with LH induced the highest increase of the CD44 receptor, followed by FSH and FSH LH treatments. These results suggest that HAS2 is mainly responsible for rapid HA synthesis in bovine COCs and GCs. In bovine COCs, the transcriptional up-regulation of both HAS2 and the receptor CD44 appear to be important prerequisites for initiating HA-mediated effects during final oocyte development and sperm-egg interaction.

An unpredictability of ovarian response still remains the major problem concerning ovine reproductive programs. The influence of several environmental, genetic, and ovarian cycle effects on oocyte/embryo yield from donor females has been previously reported. The present research has been designed to exclude aforementioned causes of variability, thus to verify embryogenic competence in homogenous groups of animals. For this purpose we used prepubertal ewes kept under identical conditions. Initially, we stimulated three groups of prepubertal ewes at various ages and used a number of gonadotropin treatments to assess differences in oocyte competence between individuals. The results revealed the repeatability of response within individual donor lambs throughout the study. Moreover, once the variability in both oocyte and embryo yield between homogenous groups of donors was revealed alongside the influence of age and type of gonadotropin treatment (P < 0.001), we investigated whether the individual donor effect persisted among genetically similar animals. Therefore, we compared oocyte and subsequent embryo output of sibling lambs derived from the most efficient donor. Here the genetic homogeneity of sisters kept under identical conditions substantially improved the uniformity of either follicular response or embryo production, suggesting that the genotype plays a primary role in establishing follicular recruitment and developmental capability of oocytes. This observation consents to predict the ovarian performance from a single ewe already in early prepuberty (i.e., to qualify the female to breeding programs).

Maternally imprinted PEG10 and SGCE, separated by ∼2.15 Mb from Syncytin (HERV-W) gene at 7q21.3, are implicated in choriocarcinoma and Silver-Russell syndrome. Here we have analyzed the temporal regulation of mRNA expression of these genes in placenta and demonstrate that Syncytin gene activation is highest in term placenta, PEG10, downregulated at early hypoxic phase, and highly activated at 11–12 wk of gestation. In contrast, transcription from SGCE remained unchanged throughout pregnancy, suggesting two neighboring imprinted genes are differentially regulated at very early pregnancy. Additionally, accumulation of two major species of mRNA (8 kb and 3.1 kb) encoded by HERV-W in placenta is regulated: 3.1 kb mRNA level remained unchanged throughout pregnancy, whereas the production of 8 kb species was highest in term placenta. Western blot and immunohistochemical staining of placental tissues with monoclonal antibodies revealed a marked reduction of syncytin glycoprotein synthesis in late pregnancy. Therefore, the relative levels of 3.1 kb and 8 kb mRNAs in trophoblasts could regulate syncytin protein synthesis, possibly by competition of the two mRNA species for translational apparatus.

The expression pattern of EP2 variants was examined in the rhesus monkey (Macaca mulatta). Using reverse transcriptase-polymerase chain reaction and rapid amplification of complementary cDNA protocols, 11 message variants were identified in rhesus epididymis, only three of which (EP2B, EP2C, and EP2E) have previously been reported. The most abundant variant found in human, EP2A, was not found in rhesus. Seven of the eight new rhesus EP2 variants (EP2J–EP2Q) use previously unidentified 5′-splicing sites in exon 3, and four variants use three previously unidentified exons whose counterparts are present in the human EP2 gene. Overall, 3 of the 11 variants, EP2C, EP2E, and EP2Q, code for β-defensin-like peptides whose probable physiological role is to protect the male reproductive tract against microbial invasions. Because of the complex splicing pattern that causes some downstream exons to be read in any of the three reading frames, the N-termini of the other eight EP2 peptide variants consist of a partial β-defensin motif with three cysteines, followed by amino acid sequences that have no recognizable homology to known proteins.

Previously, we reported that cloned embryos derived from an immortalized bovine mammary epithelial cell line (MECL) failed to develop beyond 12- to 16-cell stage. To analyze whether induction of a senescent-like phenotype in MECL can improve their ability to support the development after transfer into enucleated oocytes, we treated MECL with DNA methylation inhibitor 5-aza-2-deoxycytidine (Aza-C), histone deacetylase inhibitors trichostatin A (TSA), sodium butyrate (NaBu), or 5-bromodeoxyuridine and used those cells for nuclear transfer. Primary bovine fetal fibroblasts (BFF) were used as control. All agents were capable to induce features of senescence including reduced cell proliferation, enlarged cell size with a considerable proportion of cells stained positive for acidic senescence-associated β-galactosidase and G1/S cell cycle boundary arrest in MECL. Aza-C treatment induced genome demethylation. Acetylation of H3 and H4 was increased after TSA treatment in both MECL and BFF, whereas no obvious changes in global H3 or H4 acetylation were detected after NaBu treatment. Nuclear transfer experiments following diverse treatments demonstrated that the induced senescent-like phenotype of MECL did not confer their ability to support embryonic development, although 7.3% of reconstructed embryos derived from NaBu-treated cells developed to morula stage. Intriguingly, a much higher proportion of cloned embryos developed to blastocysts when using NaBu-treated BFF, compared with using untreated BFF (59% versus 26%). Our results suggest that the developmental failure of donor nuclei from bovine immortal cells could not be reversed by induction of senescent-like phenotype. The beneficial effect of NaBu on the developmental potential of cloned embryos reconstructed from BFF merits further studies.

Sertoli cells play a number of roles in supporting spermatogenesis, including structural organization, physical and paracrine support of germ cells, and secretion of factors necessary for germ cell development. Studies with microtubule disrupting compounds indicate that intact microtubule networks are crucial for normal spermatogenesis. However, treatment with toxicants and pharmacologic agents that target microtubules lack cell-type selectivity and may therefore elicit direct effects on germ cells, which also require microtubule-mediated activities for division and morphological transformation. To evaluate the importance of Sertoli cell microtubule-based activities for spermatogenesis, an adenoviral vector that overexpresses the microtubule nucleating protein, γ-tubulin, was used to selectively disrupt microtubule networks in Sertoli cells in vivo. γ-Tubulin overexpression was observed to cause redistribution of Sertoli cell microtubule networks, and overexpression of a γ-tubulin-enhanced green fluorescent protein fusion protein was observed to localize to the site of elongate spermatid head attachment to the seminiferous epithelium.

The degree of germ cell dependence on Sertoli cell-mediated activities has been a subject of considerable attention. Sertoli cell secretory pathways have been extensively studied both in an effort to understand their normal physiologic roles and as targets for pharmacologic and toxicant activity. To determine the degree to which normal spermatogenesis depends on key functions of the Sertoli cell microtubule network, adenoviral vectors that overexpress the microtubule nucleating protein, γ-tubulin, were delivered to Sertoli cells in vivo. γ-Tubulin overexpression disrupts the Sertoli cell microtubule network (as described in the companion article); leads to gross disorganization of the seminiferous epithelium, inducing retention of spermatids and residual bodies; and causes germ cell apoptosis. These data are consistent with earlier studies in which toxicants and pharmacologic agents were used to disrupt microtubule networks. These data confirm that Sertoli cell microtubule networks play an important role in maintaining the organization of the seminiferous epithelium and that in the absence of an intact Sertoli cell microtubule network, germ cell viability is impaired.

Labor is preceded by cervical ripening through upregulation of interleukin (IL)-1β, IL-8, and increased prostaglandin synthesis via inducible type 2 cyclooxygenase (COX-2). Progesterone maintains myometrial quiescence during pregnancy. In this study, we examined the effects of IL-1β and progesterone on IL-8 and prostaglandin E₂ (PGE₂) synthesis and IL-8 and COX-2 mRNA and promoter activity in amnion cells and lower segment fibroblast (LSF) cells. In both cell types, progesterone had no effect on basal IL-8 or PGE₂ synthesis. In LSF cells, IL-1β significantly increased IL-8 and PGE₂ synthesis and COX-2 and IL-8 mRNA expression, but progesterone significantly attenuated these effects. In prelabor amnion cells, IL-1β also increased IL-8 and PGE₂ synthesis and both COX-2 and IL-8 mRNA and promoter expression; however, progesterone significantly attenuated these effects on IL-8 and PGE₂ synthesis and COX-2 expression. In postlabor amnion cells, IL-1β increased IL-8 and PGE₂ synthesis and COX-2 expression, but progesterone did not attenuate the effect of IL-1β upon IL-8 synthesis. Progesterone repression of IL-8 and COX-2 in LSF cells suggests that IL-8 and COX-2 have similar regulatory mechanisms in LSF cells and that progesterone may play a role in maintenance of cervical competence. The lack of effect of progesterone on IL-8 in postlabor cells may be the result of downregulation of the progesterone receptor during labor.

Human CG is a pregnancy marker secreted by the placenta, and it utilizes the same receptors as does LH. Human CG is a heterodimer, and its subunits are expressed in tissues other than placenta. Similarly, LH/hCG receptors are also expressed in multiple tissues; however, the physiological significance of this expression is unknown. Free hCGβ is efficiently secreted in vitro in transfected cells and is highly expressed in many human cancers; however, the biological effects of free hCGβ in vivo are unknown. To study in vivo consequences of elevated levels of free hCGβ and hCG dimer in both male and female reproductive physiology, we used mouse metallothionein 1 promoter to generate multiple lines of transgenic mice that overexpressed either one or both subunits of hCG. Although mice expressing the glycoprotein hormone α subunit are normal and fertile, both male and female transgenic mice overexpressing only the hormone-specific hCGβ subunit are infertile. The hCGβ subunit-expressing transgenic female mice progressively develop cystic ovaries, whereas the male transgenic mice are infertile but otherwise are not phenotypically discernible. In contrast, both the male and female transgenic mice coexpressing high levels of the hCG subunits (i.e., the hCG dimer) demonstrate multiple reproductive defects. The male transgenic mice have Leydig cell hyperplasia, very high levels of serum testosterone, reduced testis size, and dramatically enlarged seminal vesicles and are infertile and display overly aggressive behavior when caged with females. The female transgenic mice are also infertile, have elevated levels of serum estradiol, and progressively develop hemorrhagic and cystic ovaries with thecal layer enlargement and stromal cell proliferation and degenerating kidneys. These results suggest that the in vivo biological effects of ectopically expressed free hCGβ subunit are distinct from those of the hCG dimer and are gender specific. These transgenic mice are useful models for studying the biology of free hCGβ subunit, for further analyzing the gain of function effects of hCG during early Leydig cell development, and for studying the roles of hCG in ovarian and kidney pathophysiology and function.

We investigated the role of endogenous redox activity in regulating the signal transduction pathway leading to tyrosine phosphorylation in mouse spermatozoa. Endogenous redox activity was monitored using a luminol-peroxidase chemiluminescent probe. Chemiluminescence increased in spermatozoa that were actively undergoing cAMP-mediated tyrosine phosphorylation events associated with capacitation and was inhibited in a dose-dependent manner by addition of catalase or diphenylene iodonium, both of which also inhibited tyrosine phosphorylation within the cell at points downstream of cAMP. Excluding bicarbonate from the incubation medium reduced the redox activity of sperm by 80–90% and dramatically reduced tyrosine phosphorylation. This study provides the first evidence that tyrosine phosphorylation associated with capacitation in mouse spermatozoa is redox regulated by a flavinoid-containing enzyme involving mediation by hydrogen peroxide. Bicarbonate regulated the redox activity of mouse spermatozoa, and this regulation may contribute to the impact of this anion on tyrosine phosphorylation during capacitation of mouse spermatozoa.

The source of yolk proteins in crustacean ovaries has been the subject of controversy for several decades, and both extraovarian and intraovarian synthesized proteins have been implicated. To offer a new insight into the relationship of vitellogenin (VTG) and vitellin (VT), a comparison of extraovarian VTG and ovarian VT of the marine shrimp Penaeus semisulcatus was performed at the protein and cDNA levels. Two cDNAs (7920 and 2068 nucleotides [nt]) were sequenced for VTG from the ovary and one cDNA (7920 nt) was sequenced from the hepatopancreas. VTG cDNA from hepatopancreas was similar to VTG cDNA from ovary. Although a VTG gene was also found in the males, ∼7.8-kilobase transcripts were only detected in the ovary and hepatopancreas of females. The mRNA expression pattern was related to the stage of ovarian development and to the molt cycle, as determined by real-time polymerase chain reaction assay. VTG and VT apoproteins were composed of two and three major subunits, respectively, as shown by SDS-PAGE. N-terminal sequences of these subunits revealed the presence of a cleavage site at a consensus motif for a subtilisin-like endoprotease in VTG and VT and an additional cleavage site in VT revealed by an unidentified endoprotease. These results indicate that penaeid shrimps constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.

Zona-binding inhibitory factor-1 (ZIF-1), a glycoprotein in human follicular fluid, reduces the binding of spermatozoa to the zona pellucida. ZIF-1 has a number of properties similar to those of glycodelin-A from human follicular fluid. The objective of this study was to compare the biochemical characteristics of these two glycoproteins. N-terminal sequencing and protease-digested peptide mapping showed that ZIF-1 and glycodelin-A have the same protein core. However, these glycoproteins differ in their oligosaccharide chains, as demonstrated by fluorophore-assisted carbohydrate electrophoresis, lectin-binding ability, and isoelectric focusing. ZIF-1 inhibited spermatozoa-zona pellucida binding slightly more than did glycodelin-A and significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. Indirect immunofluorescence staining revealed specific binding of glycodelin-A and ZIF-1 to the acrosome region of human spermatozoa, where ZIF-1 produced a stronger signal than did glycodelin-A at the same protein concentration. These data suggest that ZIF-1 is a differentially glycosylated isoform of glycodelin that potently inhibits human sperm-egg interaction. Future study on the function role of ZIF-1 would provide a better understanding of the regulation of fertilization in humans.