Accumulating evidence suggests that leptin may play important roles in preimplantation embryonic development, although this remain controversial, and little is known about whether leptin has a stage-dependent regulatory effect on development of porcine embryos derived by parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). The objective of this study was to investigate the effects of addition of leptin to in vitro culture (IVC) medium on development of porcine embryos derived by PA and SCNT. We found that addition of 50 ng/ml human recombinant leptin improved the rate of PA embryos reaching the blastocyst stage and increased the total cell number of blastocysts compared with the control group. The maximal blastocyst rate of SCNT embryos was achieved at 50 ng/ml, and the total cell number of blasocysts was increased significantly at 500 ng/ml leptin concentration. However, the ratio of the inner cell mass (ICM) to total cell number was not affected in any of the groups. Supplementation of leptin (50 ng/ml) from day 3, approximately the 4-8-cell stage, as in the case of the positive control, significantly increased the blatocyst rate of PA embryos compared with the negative control and inhibited cell apoptosis. There were no beneficial effects on embryonic development when 50 ng/ml leptin was added to the culture medium from day1 to day 3 or from day 4 to day 6. These results indicate that leptin could improve the development and the quality of PA and SCNT embryos; and 50 ng/ml leptin performs its primary stimulatory effect at 4-8-cell stage and that leptin may have no effect on the maternal-zygote transition (MZT) of porcine PA and SCNT embryos.

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The effects of long-term blockade of prolactin (PRL) action by bromocriptine (BRC) treatment on uterine carcinogenesis and on related ovarian physiology were investigated using a rat uterine cancer model. Ten-week-old cycling female Donryu rats, a high yield strain for uterine corpus tumors (endometrial adenocarcinomas), were treated with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), as a tumor initiator, and injected with 1 mg/kg body weight BRC subcutaneously 4 times per week until 14.5 months of age to block the proestrus PRL surge. The study was terminated at 15 months of age, and the results showed that long-term BRC treatment significantly inhibited endometrial adenocarcinoma development in terms of both incidence (34.6% to 13.0% with significant difference at 5%) and multiplicity (0.35 to 0.18 with significant difference at 5%), which indicates the number of adenocarcinomas per animals. While BRC did not affect estrous cyclicity in the treated animals, a significant decline was evident in the serum 17β-estradiol (E2) to progesterone (P) ratio (E: P ratio), and the serum E2 level showed a decreased tendency at 15 months of age. While the precise pathway to the inhibitory effect could not be determined; the pathway by which ovarian hormonal imbalance decreases the serum E: P ratio most likely plays a crucial role.

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To verify target organ(s) of leptin in the reproductive system of the Japanese black bear, we examined the expression of leptin receptor (Ob-R) protein in ovaries and uteri collected from July to December 2006 by immunohistochemical techniques. Eleven of 22 female Japanese black bears examined had corpora lutea (CLs) in their ovaries and were thought to have undergone or to have terminated delayed implantation in the early pregnancy stage. The CLs were classified into 3 types based on morphological features. The maximum diameters of Type 1 CLs ranged 3 to 7 mm, and the luteal cells contained numerous vacuoles in the cytoplasm, which suggests that this type of CL was functional. The maximum diameters of Type 2 CLs were approximately 7 mm, and the luteal cells contained fewer vacuoles in the cytoplasm, which suggests that this type of CL was in the early stage of regression. Finally, the maximum diameters of Type 3 CLs were approximately 1 mm, and these CLs contained collagen fibers among their luteal cells, which suggests that this type of CL had regressed. The 3 types of CLs showed different reactions to Ob-R, with positive staining in Type 1, much less positive staining in Type 2 and nearly negative staining in Type 3. In bears having CLs with functional and regressive features (Type 1 and 2 CLs), Ob-R was also immunolocalized in the developed glandular and ductal endometrial epithelium. In contrast, the Ob-R was absent in the undeveloped endometrial epithelium in bears with regressed (Type 3 CLs) or no CLs. These findings suggested that leptin directly targets CLs and the endometrium so as to develop and maintain them during the delayed implantation period in Japanese black bears.

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In contrast to those of other mammals, canine oocytes are ovulated at the germinal vesicle (GV) stage and then progress to the metaphase II (MII) stage in the oviduct. In other species, oocytes at the MII are widely used for in vitro fertilization or as recipients in somatic cell nuclear transfer. Many researchers have tried to improve the in vitro maturation (IVM) of canine oocytes. However, the proportion of MII oocytes remains low, resulting in poor efficiency of embryogenesis in vitro. This leads us to the possibility that the in vitro cytoplasmic maturation of canine oocytes is insufficient. Furthermore, the optimal culture period for IVM of canine oocytes is controversial, and physiological evaluation is required to improve canine IVM. We show here the time-dependent changes in mitogen-activated protein kinase (MAPK) and p34^cdc2 kinase activities in canine oocytes during IVM, since it is well known that both MAPK and p34^cdc2 kinase are activated following meiotic progression and show high activities in the MII stage in other species. Immediately after collection from ovaries, most oocytes were arrested at the GV stage, which was maintained until 24 h of culture. At 48 h of culture, more than half of the oocytes had progressed beyond the MI stage. A higher proportion of MII oocytes were observed with 72 h of culture compared with other culture periods. MAPK activity was found to increase in a time-dependent manner and reached a plateau at 72 h of culture. The level of p34^cdc2 kinase activity also increased in a time-dependent manner, with its maximal level observed after 72 h of culture. Activity was decreased with 96 h of culture, although there was no significant difference in the proportion of MII oocytes between 72 and 96 h. Our data thus show that the optimal culture period for IVM of canine oocytes is 72 h because both MAPK and p34^cdc2 kinase showed high activities at that time.

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Four methods of cell cycle synchronization of porcine preadipocytes for use as nuclear donors in somatic cell cloning were compared: serum starvation, differentiation induction, contact inhibition and roscovitine treatment. After three days of differentiation induction, the percentage of nuclear donor cells synchronized at the G0/G1 phase reached a peak value of 91.8%, which was significantly higher (P<0.05) than the percentage attained by serum starvation (84.9-89.8%), contact inhibition (78.3-83.7%) or roscovitine treatment (67.8-80.3%). Cell cycle synchronization by serum starvation, contact inhibition and roscovitine treatment all increased the percentage of apoptotic cells, while no increase was observed when the donor-cell cycle was synchronized by differentiation induction (Annexin V-positive: 15.7% to 19.3% vs. 7.7%, P<0.05; TUNEL-positive: 12.8% to 14.0% vs. 8.3%, P<0.05). Additionally, comparison of the in vitro development of nuclear transfer (NT) embryos formed from the nuclei of differentiation-induced or serum-starved preadipocytes revealed that, in both cases, a high proportion of embryos developed to the blastocyst stage (39.0 and 33.7%, respectively). In this study, NT embryos reconstructed with preadipocytes synchronized by differentiation induction were transferred to four recipient pigs, three of which gave birth to a total of 17 piglets (4.2%, 17/403). These results demonstrate that donor-cell cycle synchronization by differentiation induction enables effective production of cloned pigs. The findings also indicate that differentiation induction of multipotent cells is an excellent method of cell cycle synchronization that permits highly efficient synchronization of cells at the G0/G1 phase.

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The production of transgenic pigs using somatic cell nuclear transfer (scNT) has been widely described, but a technique for removing nontransfected donor cells and for creating different founder animals has not yet been fully elucidated. In this study, four different expression vectors (pBC1hEPO, pMARBC1hEPO, pBC1hEPOwpre and pMARBC1hEPOwpre) were compared to determine the highest transgene expression, ideal conditions of enrichment of recombinant cells in vitro and efficiency of transgenesis following transfection into HC11 mammary epithelial cells. The highest protein expression in HC11 cells was obtained from the pMARBC1hEPOwpre expression vector. Next, we evaluated the efficiency of transgenic pig production by using geneticin (G418) selection alone or by using real-time PCR selection following G418 selection. Ideal enrichment of recombinant cells was obtained by a combination of real-time PCR and G418 selection; embryos reconstructed using donor cells selected by a combination of real-time PCR and G418 selection gave rise to nine piglets, all of which were transgenic. Among them, three founder transgenic pigs were established. Exogenous DNA fragments were shown to be integrated into chromosomes 1q2.4, 1p2.3 and 6q2.4, respectively, in these three pigs. However, the transgenic rate using G418 selection alone was only 33% (two of six pigs) and showed a very low efficacy compared with that of the combination of real-time PCR and G418 selection. Our results provide a valuable experimental model for applying and evaluating transgenic technology in pigs.

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The effect(s) of oral calcium and vitamin D₃ were examined on the expression of duodenal and renal active calcium transport genes, i.e., calbindin-D9k (CaBP-9k) and calbindin-D28k (CaBP-28k), transient receptor potential cation channels (TRPV5 and TRPV6), Na⁺/Ca²⁺ exchanger 1 (NCX1) and plasma membrane calcium ATPase 1b (PMCA1b), in CaBP-9k KO mice. Wild-type (WT) and KO mice were provided with calcium and vitamin D₃-deficient diets for 10 weeks. The deficient diet significantly decreased body weights compared with the normal diet groups. The serum calcium concentration of the WT mice was decreased by the deficient diet but was unchanged in the KO mice. The deficient diet significantly increased duodenal transcription of CaBP-9k and TRPV6 in the WT mice, but no alteration was observed in the KO mice. In the kidney, the deficient diet significantly increased renal transcripts of CaBP-9k, TRPV6, PMCA1b, CaBP-28k and TRPV5 in the WT mice but did not alter calcium-relating genes in the KO mice. Two potential mediators of calcium-processing genes, vitamin D receptor (VDR) and parathyroid hormone receptor (PTHR), have been suggested to be useful for elucidating these differential regulations in the calcium-related genes of the KO mice. Expression of VDR was not significantly affected by diet or the KO mutation. Renal PTHR mRNA levels were reduced by the diet, and reduced expression was also seen in the KO mice given the normal diet. Taken together, these results suggest that the active calcium transporting genes in KO mice may have resistance to the deficiency diet of calcium and vitamin D₃.

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In sika deer, the normal method of estimating fetal age, based on fetal weight, is not applicable during the early pregnancy period. The objective of the present study was to describe the growth and development of sika deer fetuses and to establish a method for fetal age estimation during early pregnancy using ultrasonography. Five captive female Hokkaido sika deer (Cervus nippon yesoensis) were observed for estrus and mated (day 0) with an intact male. At two- or three-day intervals, fetuses were observed by rectal ultrasonographic scans until 59-61 days of gestation. The straight crown-rump length (SCRL), curved crown-rump length (CCRL), head length (HL), trunk depth (TD) and heart rate (HR) of the fetuses were measured. Linear regression equations were computed for each measurement together with fetal age. Analyses were conducted after transformation to a natural logarithm for SCRL and CCRL. All equations were significant (P<0.001), with SCRL becoming measurable earlier (day 20) than the others and yielding the best correlation (Days= -2.08+14.15 LnX: X=SCRL, Ln=natural logarithm). Therefore, we concluded that a precise estimation of fetal age in early gestation is best performed using SCRL measurements.

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To examine whether oxygen (O₂) and nitric oxide (NO) are temporally associated with the acute changes in luteal function during luteolysis, we determined the real-time changes in the circulating concentrations of progesterone (P4) and nitrite/nitrate (the stable metabolites of NO) and the partial pressure of oxygen (pO₂) during prostaglandin F_2α (PGF_2α)-induced luteolysis in cattle. Catheters for frequent blood sample collection were inserted into the ovarian vein (OV), jugular vein (JV) and aorta abdominalis (AA) in 12 cows on Day 9 of the oestrous cycle (oestrus=Day 0). On Day 10, the cows were randomly divided into two groups and treated with a luteolytic dose of a PGF_2α analogue or saline solution (control). Blood samples were collected at -2, -1, 0, 0.25, 0.5, 0.75, 1 and 2 h and then at 2-h intervals until 12 h after treatment (0 h). Injection of a PGF_2α induced a significant decrease in the concentrations of P4 in OV plasma within 2 h. The decrease in P4 concentrations was preceded by an increase in the NO concentrations in the blood collected from OV, JV and AA. Basal pO₂ was significantly higher in OV blood than in JV blood (P<0.05). PGF_2α injection increased pO₂ in OV blood between 0.5 and 2 h. These results demonstrate that PGF_2α induced an acute increase in pO₂ and NO in the ovarian circulation and suggest that O₂ and NO are involved in the early events of CL regression, including inhibition of P4 secretion and output, in cattle.

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In the mouse, ADAM3, a well-characterized testis-specific protein of the A disintegrin and metalloprotease (ADAM) family, has a crucial role in fertilization by mediating sperm binding to the egg zona pellucida. However, little is known about ADAM3 in other species, such as domestic pigs. We have identified porcine ADAM3 and analyzed the protein. RT-PCR and trypsinization of sperm surface proteins revealed that porcine ADAM3 is expressed at high levels in the testis and on the sperm surface. Furthermore, an IVF inhibition assay with a recombinant porcine ADAM3 disintegrin domain showed that treatment of the disintegrin domain effectively prevented pig sperm-egg interactions. In the present study, we demonstrated the presence of ADAM3a and ADAM3b molecules in the pig and examined their roles in fertilization.

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The objective of this study was to clarify the effects of a second protocol of ovulation synchronisation starting on day 26 after timed artificial insemination on ovarian profile and days open in dairy cows diagnosed as nonpregnant. Ninety-four Holstein-Friesian cows received intramuscular injections of a GnRH analogue (GnRH), 100 μg fertirelin, on day 0 and a prostaglandin F_2α analogue (PG), 5 mg etyprostontromethamine, on day 7. GnRH was again administered 48 h after the PG injection, and timed artificial insemination was performed 16 to 20 h later (Ovsynch/TAI). Twenty-six of the 94 cows returned to oestrus within 26 days after TAI and were inseminated. Of the other 68 cows, 44 were not pregnant and were randomly allocated to undergo another Ovsynch/TAI protocol (Resynch group; n=23) or AI only after detection of oestrus (Control group; n=21). The ovarian and hormonal profiles were compared between the first and second Ovsynch protocol periods in the Resynch group. The diameter of the dominant follicle and plasma oestradiol-17β concentration at the second GnRH injection were significantly greater than those at PG injection during the second Ovsynch period. Ovulation was synchronised in all of the animals in the second Ovsynch period. The AI submission rates, mean AI intervals and pregnancy rates of the Resynch and Control groups were 100% and 57.1%, 36.0 ± 0.0 and 43.2 ± 10.9 and 30.4% and 14.3%, respectively. The mean AI interval was 7 days shorter and the pregnancy rate was higher in the Resynch group than in the Control group, although no significant differences were found due to the small number of the animals. In conclusion, the Resynch protocol initiated on day 26 after TAI in the first protocol has the potential to reduce days open and increase the pregnancy rate in dairy cows.

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We studied the secretory function of the corpus luteum (CL) in cows following different estrus synchronization protocols. Estrus was synchronized using one (n=4) or two injections (n=5) of prostaglandin F_2α (PGF_2α; dinoprost), two injections of different analogues of PGF_2α (aPGF_2α), luprostiol (n=5) and cloprostenol (n=5), at eleven-day intervals, a gestagen implant (norgestomet, n=5, for 10 days) or norgestomet together with a subsequent dinoprost injection on the day of implant removal (n=5). CL samples were collected by ovariectomy on Day 7-8 of the estrous cycle. Luteal strips were stimulated with LH (100 ng/ml) or prostaglandin E₂ (PGE₂, 10^-6M) for 24 h in culture media. The progesterone (P₄) and PGE₂ concentrations in the media were measured by enzyme immunoassay. In the control CL (spontaneous estrus; n=5), LH and PGE₂ stimulated P₄ and PGE₂ (P<0.001). The effects of both factors on P₄ were reduced in the CL following dinoprost- and cloprostenol-synchronized estrus (P<0.05) and were absent in the luprostiol-synchronized CL (P>0.05). In the norgestomet-synchronized CL, the stimulatory effects of LH and PGE₂ were higher compared with the CL synchronized by aPGF_2α (P<0.05). Pharmacological manipulation of the estrous cycle using aPGF_2α may cause lower P₄ secretion. Estrus synchronization inhibited CL sensitivity to luteotropic factors. Therefore, attention should be focused on the estrous synchronization method in both in vivo and in vitro studies of CL functions in cattle.

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Glycerol plays multi-functional roles in cellular physiology. Other than forming the backbone molecule for glycerophospholipid and triglyceride (TG), glycerol acts as an energy substrate for glycolysis. Spermatozoa are known to utilize glycerol for energy production, but there are no reports of this in oocytes. In this study, the value of glycerol as an energy substrate for bovine oocyte maturation (Exp. 1) and the gene expression of glycerol kinase (GK), an enzyme crucial for cellular glycerol utilization, in bovine oocytes and early embryos (Exp. 2) were examined. In Exp. 1, in vitro maturation (IVM) was conducted using synthetic oviduct fluid supplemented with/without glucose (1.5 mM) and/or glycerol (1.0 mM), and maturation rate, degree of cumulus expansion, glucose consumption and lactate production by cumulus-oocyte complexes (COC) were examined. In Exp. 2, to examine the developmental expression of GK mRNA, cumulus cells, oocytes and embryos at the 2-, 8- and 16-cell, morula, expanded blastocyst and hatched blastocyst stages were obtained in separate experiments, and the expression of GK mRNA was quantified using a real-time PCR. Glycerol did not support oocyte maturation or cumulus expansion. Addition of glycerol to glucose-supplemented media significantly decreased the maturation rate. Expression of GK mRNA was very low in cumulus cells, whereas an appreciable level of the transcript was observed in the oocytes. GK mRNA was detected in embryos at all the stages examined, and its expression significantly increased at the morula stage. These results indicate that glycerol, at least at the present concentration, is not beneficial as a constituent of the medium for bovine oocyte maturation. However, the appreciable levels of GK mRNA found in the oocyte and embryo imply a physiological role for glycerol in bovine oocyte maturation and embryo development.

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The migratory ability of chick primordial germ cells (PGCs) transferred into quail embryos was investigated. One, ten, twenty, fifty or one hundred chick PGCs were transferred into the dorsal aorta of 2.5-day-old quail embryos. One day later, the embryos were isolated, and serial sections were prepared after embedding in paraffin. The sections were then double-stained with periodic acid-Schiff (PAS) and hematoxylin, and the numbers of PAS-positive chick PGCs in the germinal epithelium, gonadal area, head area and trunk area of the embryos were determined. Approximately 70% of the PGCs were detected in the embryos 1 day after transfer, with roughly 60% in the gonadal region and 10% in the extragonadal region. This ratio was consistent regardless of the number of PGCs transferred into the embryos. These data suggest that migration of chick PGCs into the gonadal and extragonadal regions of the quail embryo occurs probabilistically regardless of the number of chick PGCs transferred into quail embryos.

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The present study was undertaken to determine the effect of nucleosides on nuclear and cytoplasmic maturation of mouse oocytes. Oocyte-cumulus complexes (OCCs) were collected from large antral follicles 4 h after eCG-hCG treatment and cultured in maturation medium with or without nucleosides (4 ribo- and 4 deoxyribonucleosides) for 12 h. A majority of the oocytes examined developed to the metaphase-II stage, and the same result was found with in-vivo matured oocytes. However, mitochondrial membrane potential (MMP) was significantly lower in the oocytes matured in the presence of nucleosides than in the nucleoside-free controls. Oocyte MMP increased in vivo between 8 to 12 h after hCG injection, whereas no increases in MMP were observed in oocytes matured in the presence of nucleosides. Oocyte MMP was significantly lower only when OCCs were exposed to nucleosides for the latter 8 h of IVM. When OCCs were exposed to adenosine during the latter 8 h of IVM, MMP was lower compared with in-vivo matured oocytes, whereas a mixture of other ribonucleosides (guanosine, cytidine and uridine) did not affect the level of MMP. The developmental competence of oocytes exposed to adenosine during the latter 8 h of IVM was lower after parthenogenetic activation due to the lower pronuclear formation of the oocytes. These observations indicate that adenosine inhibits increases in oocyte MMP during the latter half of meiotic maturation and detrimentally affects cytoplasmic maturation.

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The objectives of this study were to obtain morphological data for normal spermatozoa and to investigate seasonal changes (the early, mid- and post-mating seasons) in abnormal morphology of spermatozoa and the characteristics of semen in Japanese black bears. Semen was collected by electroejaculation from 34 captive male Japanese black bears a total of 74 times. Length of head, width of head, length of midpiece and total length of the spermatozoa were 6.3 ± 0.4, 4.5 ± 0.3, 10.4 ± 0.7 and 69.6 ± 3.1 μm (mean ± SD; 20 semen, 200 spermatozoa), respectively. In the semen collected during the mid-mating season, ejaculate volume, ejaculate pH, sperm concentration, total sperm count, motility, viability and intact acrosomes were 0.46 ± 0.36 ml, 7.3 ± 0.4, 659 ± 644 × 10⁶/ml, 214 ± 208 × 10⁶, 82.9 ± 9.6%, 89.3 ± 9.5% and 97.0 ± 3.2% (mean ± SD; n=21, in ejaculate pH n=8), respectively. Sperm motility and viability in the early (n=7) and mid-mating (n=21) seasons were significantly higher than in the post-mating (n=8) season. The rates of detached heads in the early and mid-mating season were significantly lower than in the post-mating season. The main abnormal morphologies observed (mean ± SD%; n=23) were simply bent tail (19.9 ± 22.6), distal droplets (13.5 ± 11.7), proximal droplets (9.6 ± 7.8), teratoid spermatozoa (6.7 ± 10.7), knobbed acrosome (4.9 ± 8.6), acrosome damage (3.7 ± 2.8) and bent midpiece (3.7 ± 5.1). The data will be useful for artificial breeding and further research on male reproductive physiology in this species.

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Although the fallopian tube provides a sufficient environment for fertilization and early embryo development, the mechanism by which it does this is unclear. It is known that the transforming growth factorβ (TGFβ) superfamily plays important roles in various reproductive functions. Betaglycan, originally characterized as a TGFβ type III receptor lacking a clearly identifiable signaling motif, has been shown to be important for the high-affinity binding of TGFβs to the type II receptor. To our knowledge, there has been no study showing expression of betaglycan in the rat oviduct. Therefore, in this study, we examined the distribution of betaglycan in various rat tissues and its expression patterns in the oviduct during the estrous cycle. Northern blot analysis of various rat tissues showed that the adrenal gland, ovary and oviduct contained abundant amounts of 6.4-kb betaglycan mRNA. Furthermore, the mRNA level of betaglycan was highest after the LH surge that induced ovulation. The betaglycan protein, detected using immunohistochemistry, was especially abundant in the epithelium of the oviduct. Furthermore, in pregnant mare serum gonadotropin (PMSG) primed-rats, the expression of betaglycan was increased significantly by stimulation with human chorionic gonadotropin (hCG). RT-PCR analysis showed co-localization of other TGFβ family receptors (TGFβ types I and II, activin receptor types Ia and Ib and activin receptor types IIa and IIb) with betaglycan in the oviduct. Since betaglycan along with other TGFβ family receptors is abundantly expressed in the epithelium of the oviduct and its expression changes during estrous, it may also play an important role in the oviduct.

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Growth hormone (GH) plays a pivotal role in the regulation of growth, development and body composition. In order to provide new insights into estrogenic endocrine disruptor (ED) activities in the pituitary gland and the potential role played by estrogen receptors (ERs) in mediating their effects in vivo, we examined GH expression in the pituitary gland of an immature rat model. At postnatal day 14, immature rats were treated with various doses of 4-tert-octylphenol (OP), p-nonylphenol (NP) and bisphenol A (BPA), and the GH mRNA and protein expression levels were analyzed by real-time quantitative PCR and western blot/immunohistochemistry (IHC), respectively. An anti-estrogen (ICI 182780) was used to examine the potential involvement of ERs in ED-induced GH expression during critical windows of development. GH mRNA expression increased significantly 48 h after treatment with a high dose (600 mg/kg body weight [BW]) of OP or NP. However, this induction was abolished completely by co-treatment with ICI 182780. No significant difference in GH mRNA expression was observed following treatment with BPA or co-treatment of BPA with the anti-estrogen. Exposure to high doses (600 mg/kg BW) of these EDs significantly enhanced GH protein expression in the rat pituitary gland, whereas pretreatment with ICI 182780 markedly reduced this expression. Taken together, we have demonstrated for the first time that in vivo exposure to EDs can induce GH mRNA and protein expression in the rat pituitary gland and that their activities may involve an ER-mediated signaling pathway. These results may provide critical evidence for ED-induced dysregulation of pituitary GH expression and thus may be important for elucidating the potential impacts of EDs in altered body growth and development and for predicting the health risks of ED exposure in humans and wildlife.

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The dead end gene, coding an RNA binding protein, is predominantly expressed in the germ cells of vertebrates. Recently, we cloned chicken dead end homologue (CDH) and showed that expression of CDH mRNA is highly specific to primordial germ cells (PGCs) at early embryonic stages. To date, the subcelluler localization of Dead end protein in germ cell has been largely unknown due to lack of an antibody. Here, we raised a polyclonal antibody against chicken dead end homologue (CDH) to elucidate its subcellular localization in the germ cells. For comparative studies with CDH, a polyclonal antibody against chicken vasa homologue (CVH), a well-known germ cell marker, was also raised. Immunoblotting analysis for CDH protein showed a single band with a molecular size of approximately 60 kDa in the ovarian and testicular proteins. Immunofluorescence studies revealed that CDH protein was exclusively localized in the nuclei of primordial germ cells (PGCs) and germ cells at later stages, while CVH was localized in the cytoplasm. Interestingly, the germ cells distributed at the basal sides of seminiferous epithelia, such as spermatogonia, were strongly positive to CDH protein. The current study provides novel evidence that CDH is a nucleoprotein of germ cells, including PGCs.

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A simple and clear means to identify the physiological status of follicles is essential for study of follicular biology. In the present study, we verified a novel classification procedure based on analysis of the follicular population and glucose concentration in follicular fluid (FF) as an alternative method to classify bovine follicles. Paired ovaries were collected from heifers, and the number of follicles and stage of the CL were recorded. Follicles were initially divided into the following 3 groups according to diameter and the ratio of E2 and P4 (E/P): E2 active (E-A: E/P≥1), E2 inactive (E-I: E/P<1, ≥8.5 mm) and small follicles (E/P<1, <8.5 mm). E-A follicles were easily identified as E2-rich dominant follicles and were further classified according to diameter and stage of the CL as early dominant (EDF: <8.5 mm), dominant (DF: ≥8.5 mm, stages I-III) or preovulatory follicles (POF: ≥8.5 mm, stage IV). E-I follicles were classified as follows based on the status of the accompanying follicles: early atretic (EAF: without an E-A follicle), mid-atretic (MAF: with an EDF or DF) and late atretic follicles (LAF: with an EAF or POF). The follicular P4 concentrations of the MAF and LAF were significantly higher compared with that of the EAF, while follicular glucose concentration of the LAF was lower compared with those of EAF and MAF, indicating that this classification can be used to distinguish early atretic follicles from more advanced atretic follicles. Small follicles were classified as growing (GF: without E-A follicles) and suppressed small follicles (SSF: with E-A follicles). The SSF was easily identifiable by this procedure, but some GF populations likely contained SSF. To identify true GF, the ratio of E2 in the GF and accompanying EAF may be used. In conclusion, analysis of the follicular population in conjunction with biochemical indices such as steroid and glucose concentrations in FF provides a simple and accurate means of classifying bovine follicles.

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