エゾシカ (Cervus nippon yesoensis) の受胎と妊娠維持における繁殖生理学的特徴の解明

There are many species of boreal or temperate deer (Lincoln 1992) and they are short-day breeders (Sadleir 1987, Lincoln 1985, Loudon and Brinklow 1992) mating in autumn and fawning in early summer (Kaji 1988, Koizumi 1991, Matsuura et al. 2004a). Their seasonal reproductive pattern ensures that fawns are born at a time of year when food is available for lactating and the weather is favorable for survival of offspring (Loudon and Brinklow 1992). Although most of parturition takes place during period of about one month (Guinness et al. 1978a, Koizumi 1991, Birgersson and Ekvall 1997, Bowyer et al. 1998), range of 83 to 135 days for birth date due to late parturitions were reported in sika deer (Koizumi 1991, Matsuura 2004). Late conception leads to late parturition (Matsuura et al. 2004a) and late parturition results in decline in reproductive success of females, defined as the number of fawns a mother reared to one year old over a specified period of time (red deer: Clutton-Brock et al. 1982), due to increase in mortality rate of fawn (Guinness et al. 1978b) or decrease in female conception rate in the following mating season (Clutton-Brock et al. 1983). Therefore, conceptions at the appropriate breeding season and subsequent maintenance of pregnancy throughout gestation are required for females to enhance their reproductive success. Since most of female conceive at the first estrus of the season (Matsuura et al. 2004a), the change in physiological condition from the anestrous season to the estrous season is important factors which responsible for determining the time of conception. However, there is no insight into mechanism for the successful conception and physiological factor influences to the conception date, and even basic information about the reproductive physiology such as ovarian dynamics and changes in hormones around the conception are not well known in sika deer. Therefore this study was conducted to clarify the characteristics of reproductive physiology of sika deer with special interest in around conception and gestation period by revealing the changes in reproductive organs and steroid hormones. There are two approaches to study about the issue; using carcasses and using live animals. Carcasses are available from nuisance control, sport hunting and hunting for the research purpose, and provide information on morphology and histology of reproductive organs in which animals are needed to be killed. Although abundant carcasses can be obtained, information from carcasses reflects the condition only at the time they were sampled. On the other hand, studying live animals provides the temporal changes in reproductive physiology in animals. However, number of captive deer available for physiological study is limited due to lack of institution holding animals, insufficient equipment, and financial problem in Japan. Therefore, combination of these two approaches is essential for further improvement for understanding reproductive physiology of sika deer. Carcasses provide the physiological data such as occurrence of ovulation (Suzuki and Ohtaishi 1993) and steroidogenic ability of corpus luteum (CL; Matsuura et al. 2004c). In the study using carcasses, the reproductive status must be estimated from the condition of the female, and when they are pregnant, gestational ages are estimate from fetal weight in sika deer (Suzuki et al. 1996). However, present method of gestational age estimation will contain an intrinsic error at early gestational stage. The accurate estimation of early gestational age of the samples contributes to revealing characteristics of reproductive physiology around early pregnancy. Study using live animals is advantageous in understanding temporal changes in the individuals and noninvasive examination, such as behavioral observation and fecal progesterone analysis (Matsuura et al. 2004a), provided the important insight in sika deer. However, data of noninvasive examination are limited to indirect changes in reproductive physiology. To know the characteristics around the first estrus and conception more in detail, invasive methods as it has be done in other cervid species, such as blood collection for hormone assay (Plotka et al. 1977, Kelly et al. 1982, Adam et al. 1985, Garcia et al. 2003) and transrectal ultrasonography for follicular and luteal dynamics (Asher et al. 1997, McCorkell et al. 2004, 2006, 2007) of captive animals under restrain or immobilization are also needed. In sika deer, there is a unique reproductive characteristics observed from early pregnancy. It is multiple CLs formation in spite of having singleton (Yamauchi et al. 1984, Suzuki et al. 1992, Suzuki and Ohtaishi 1993). Since both CLs have ability to synthesis progesterone, it is assumed that forming surplus CL have an important role for establishment and maintenance of pregnancy (Matsuura et al. 2004c). However, significance and function of surplus CL is not obvious, and even the origin and the timing of formation of surplus CL are not known. For understanding the conception and maintenance of pregnancy in this species, these questions must be revealed. In present thesis, the author focused on conception and pregnancy period in sika deer. In chapter 1, the temporal ovarian dynamics and changes in steroid hormones were investigated from anestrous to estrous season to know the characteristics during the seasonal transition and to discuss the factor influence to the successful conception. In chapter 2, the temporal ovarian dynamics and changes in steroid hormone during conception and early gestation were investigated and the significance of multiple CLs was discussed. In chapter 3, description of fetal development and estimation of fetal age during early pregnancy was reported. In chapter 4, distribution of steroid hormone receptors in uteri derived from the wild deer were examined to know the steroid hormone action site at the each stage of pregnancy.受胎日の遅延はシカ類の繁殖成功度に悪影響を与える。そのため、適切な時期に受胎し、妊娠を維持することは繁殖成功を高める重要な要因となる。そこで本研究では、受胎時期と妊娠維持の時期に注目した。ニホンジカにおいては、卵巣動態やホルモン濃度の変化などの基本的な繁殖生理学的情報が欠けているため、どのような生理的要因が受胎時期に影響を与えているかは不明である。そこで本研究では、受胎時期と妊娠維持の時期周辺の基礎的な生理状態および生理機構の特徴について検討した。第1章では、非発情期から発情期への移行期における卵巣動態とホルモン濃度変化の特徴を明らかにすることを目的とした。8頭の飼育個体を用い、卵胞や黄体の動態、ステロイドホルモン濃度の変化を初回発情周期中とその前後の期間で比較した。発情開始以前から初回発情までは7個体を、初回発情から発情周期3～6回分の期間は3個体を実験に用いた。2～3日間隔で経直腸による超音波画像診断と血中プロジェステロンおよびエストラジオール17.濃度測定を行った。その結果、全ての個体において初回の発情以前に発情を伴わない排卵がみられた。また、それに続く一過性の黄体形成と、低濃度のプロジェステロン濃度の上昇が1回以上観察された。プロジェステロンは発情を引き起こす因子であるため、一過性のプロジェステロンによる感作は、発情期の開始に寄与していると推察された。また、発情周期中には主に2ないし3回の卵胞発育波が観察された。さらに、発情時には子宮頸管粘液の結晶化が観察され、発情の指標となることが示唆された。第2章では、受胎時期と妊娠初期における卵巣動態とホルモン濃度変化の特徴を明らかにし、妊娠しているニホンジカにおいて特徴的に見られる複数の黄体の起源と形成時期を明らかにし、その存在意義を検討することを目的とした。6頭の飼育個体を用いて受胎から妊娠初期までの卵胞と黄体の動態およびステロイドホルモン濃度の変化を2～3日間隔で調べた。その結果、初回の発情で妊娠した個体3頭において2つの黄体が形成された。2つ目の黄体は、発情に伴う排卵の後に出現した最初の卵胞発育波の主席卵胞が起源であり、これがエストロジェンの上昇と共に排卵し、黄体を形成した。一方、2回目の発情で妊娠した個体３頭では、発情後の主席卵胞は排卵せず、黄体は1つしか形成されなかった。そのため、2つ目の黄体は発情期初回の発情で受胎した場合に形成されることが示唆された。また、黄体の数によってプロジェステロン濃度に差が認められなかったため、2つ目の黄体は1つ目の黄体のみでは不十分なプロジェステロンの分泌を補助することで、初回発情での妊娠の維持に寄与していると推察された。第3章では、妊娠初期における胎子の成長を明らかにし、正確な胎齢推定を可能にすることを目的とした。5頭の飼育個体を用い、妊娠59～61日まで2～3日間隔で経直腸超音波画像診断により胎子の成長を観察した。妊娠20～26日までに、全ての個体において胎子が確認された。胎子の直頭殿長、曲頭殿長、頭長、胸深、心拍数を計測した結果、両頭殿長の対数を取った値とその他の計測値は直線的な増加を示した。胎齢推定式を算出した結果、直頭殿長が最も早期から計測可能で、高い相関を示すことが明らかとなった。したがって、直頭殿長を計測することで、正確に妊娠初期の胎齢が推定できることが判明した。第4章では、妊娠期間中の子宮におけるプロジェステロンとエストロジェンの作用部位の変化を推定することを目的とした。複数黄体を持つ24の死体から得られた子宮組織を用いて、発情直後から妊娠末期までのエストロジェン受容体. (ER.)とプロジェステロン受容体(PR)の発現を免疫組織化学的に調べた。胎齢は第3章の結果と既存の方法をもとに推定した。その結果、ER.およびPRが妊娠25日まで確認され、他の家畜反芻動物と比較すると、受胎後も遅い時期まで発現していることが明らかとなった。したがって、ニホンジカでは妊娠25日程度まで、プロジェステロンのみならずエストロジェンが作用していることが示唆された。本研究では、非発情期から発情期、受胎から妊娠初期までの卵胞や黄体の動態と血中ステロイドホルモン変化、さらに妊娠個体の子宮におけるステロイドホルモン受容体の分布から、これまで情報の少なかったニホンジカの発情期への移行期、受胎時期や妊娠初期における繁殖生理学的特徴を明らかにした。また、妊娠初期の正確な胎齢推定の方法を確立したことにより、今後野外で得られたサンプルの有効活用を可能にした

Effect of a tranquilizer on stress control in birds under restraint

Birds are easily disabled by stress, which often has adverse effects on their health, thus birds should be handled as little as possible to minimize stress. In the present study, the effects of the tranquilizer Diazepam against restraint stress were evaluated using the Japanese Quail Coturnix japonica. When restraint stress was applied to the birds by squeezing their cage, the plasma corticosterone (hormone which increases under stress) levels increased markedly, while no difference was observed in the birds that had only undergone blood sampling. Thus, the squeezed cage is a suitable instrument for examining restraint stress. Intramuscular injection of diazepam before restraint stress resulted in no significant increase in the corticosterone level. In contrast, the corticosterone level increased markedly when saline was administrered as a placebo. These findings demonstrate that the use of a tranquilizer is very effective in reducing stress, and that tranquilizers will be useful in the study of stress reduction or in the investigation of the mechanisms of stress in birds

Endometrial epidermal growth factor profile and its abnormalities in dairy cows

Normal cows have 2 peaks in endometrial epidermal growth factor (EGF) concentrations on Days 2-4 and 13-14, and the absence of peaks has been linked to reduced fertility in repeat breeder (RB) cows. However, the timing of the peaks (i.e., day of examinations) was estimated for a few cows per cycle day. Therefore, the present study characterized EGF peaks and examined if the absence of peaks in RB cows indicate either peak loss or changed timing. In Study 1, 20 Holstein cows were examined for EGF concentrations between Days 1 and 6 using repeated biopsy of the uterine endometrial tissues. Sixteen cows exhibited increased EGF concentrations for 2-3 days between Days 2 and 5. All 16 cows exhibited increased EGF concentrations on Day 3. In Study 2, 10 cows were examined for EGF concentrations between Days 11 and 16. Increased EGF concentrations for 2-3 days were found in 7 cows between Days 12 and 15. All 7 cows exhibited increased EGF concentrations on Days 13 and 14. In Study 3, 12 RB cows were examined for endometrial EGF concentrations between Days 1 and 6. Four cows exhibited an increase of EGF concentrations on Days 3 and 4, whereas 8 cows (66.7%) exhibited low EGF concentrations throughout the study period. In conclusion, Days 3 and 13-14 are suitable days to examine a cyclic change of endometrial EGF concentrations. Further, low EGF concentrations on Day 3 in RB cows indicated an absence, but not altered timing, of the EGF peak

Fertilizability of oocytes derived from Holstein cows having different antral follicle counts in ovaries

In this study, to clarify the relationship between ovarian reserve and oocyte quality, cumulus-oocyte complexes (COCs) were collected repeatedly by ovum pick-up (OPU) from cows with high and low antral follicle counts (AFCs) at short (3–4 days) and long (7 days) intervals, and COC morphologies and oocyte fertilizability were examined. The relationship between AFC and follicular growth after OPU was also investigated. Cows showing AFC of ≥30 in at least one OPU session were grouped into the high-AFC group. At a short interval, follicular sizes and COC morphologies were similar between the different AFC groups. However, the normal fertilization rate was higher in the high-AFC group than in the low one, although total penetration rates were similar. At a long interval, the percentage of COCs with poor morphology in the high-AFC group was higher and the normal fertilization rate was lower than in the low one. In the low-AFC group, normal fertilization rates at short and long intervals were similar, and mean follicular size became larger at a long than at a short interval. However, mean follicular sizes at short- and long-interval OPU were similar in the high-AFC group. In conclusion, it is suggested that oocytes derived from cows with high AFC had higher fertilizability than those from cows with low AFC when OPUs were performed at a short (3–4 days) interval. However, oocyte quality in high-AFC cows was impaired by long-interval (7 days) OPU, possibly due to the degradation of follicles

Relationship between in vitro growth of bovine oocytes and steroidogenesis of granulosa cells cultured in medium supplemented with bone morphogenetic protein-4 and follicle stimulating hormone

Bone morphogenetic protein-4 (BMP-4) and FSH play important regulatory roles in follicular growth and steroidogenesis in vivo. The purpose of this study was to investigate the effects of BMP-4 and FSH on in vitro growth (IVG) and steroidogenesis of bovine oocyte-cumulus-granulosa complexes (OCGCs). We cultured OCGCs collected from early antral follicles (0.5-1 mm) in medium without BMP-4 and FSH for 4 days and investigated the appearance of OCGCs and their steroidogenesis. During the first 4 days of IVG, morphologically normal OCGCs produced more estradiol-17 beta (E-2), but less progesterone (P-4). Morphologically normal OCGCs were subjected to an additional culture in medium supplemented with BMP-4 (0, 10, and 50 ng/mL) and FSH (0 and 0.5 ng/mL) until day 12. We examined the viability and steroidogenesis of OCGCs after 8 and 12 days of culture. Oocyte growth, characteristics of granulosa cells, and the maturational competence of oocytes were also investigated. On day 8, the viability of OCGCs cultured without FSH was higher in the 10 ng/mL BMP-4 group than in the 50 ng/mL BMP-4 group (P < 0.05). No significant difference was observed in the viability of groups cultured with FSH, regardless of the addition of BMP-4, and FSH improved the viability of 50 ng/mL BMP-4 group similar to 10 ng/mL BMP-4 group. The total number of granulosa cells was larger in 10 ng/mL BMP-4 group cultured with FSH than in 50 ng/mL BMP-4 group cultured with FSH on day 8 (P < 0.05). E-2 production decreased from days 8-12, and P-4 production increased throughout IVG culture, regardless of the addition of BMP-4 and FSH (P < 0.05). No significant differences in E-2 production were observed between groups from days 4-8, regardless of whether BMP-4 was added without FSH; however, E-2 production in the group cultured with 50 ng/mL BMP-4 was suppressed by FSH. BMP-4 suppressed E-2 production from days 8-12, regardless of whether FSH was added. The group cultured with 10 ng/mL BMP-4 without FSH showed the lowest P-4 production among all groups for all culture periods. OCGCs that produced mature oocytes tended to secrete more E-2 and less P-4 than OCGCs that produced immature oocytes. In conclusion, until day 8 of the NG culture, P-4 production by OCGCs was suppressed by the addition of 10 ng/mL BMP-4 in the absence of FSH, without inhibiting E-2 production. These conditions appear to mimic growing follicles until day 8 and mimic degenerating follicles from days 8-12 of culture

Identification of bovine seminal plasma proteins with an activity to normalize endometrial epidermal growth factor concentrations in repeat breeder cows

Epidermal growth factor (EGF) concentrations in the bovine uterus show two peaks on Days 2-4 and 13-14 during the estrous cycle in normal cows; however, these peaks were not found in about 70% of Holstein repeat breeder cows. We have demonstrated the effect of seminal plasma (SP) to normalize the endometrial EGF concentrations on Day 3 and restore fertility in repeat breeder cows. The objective of this study was to identify SP protein(s) with the activity of normalizing the endometrial EGF concentrations. Semen was collected from 5 Holstein bulls and pooled SP obtained from 30 ejaculates were used for this study. The SP protein were separated by gel filtration and 2-dimensional electrophoresis. SP fractions with molecular weight of 16-29 kDa and pI5.8-7.0 showed an activity to normalize the endometrial EGF concentrations on Day 3. Then, protein spots in these area on electrophoresis gels were extracted and subjected to liquid chromatography with tandem mass spectrometry analysis. Twelve protein spots that include four spots of osteopontin (OPN) with different molecular weights and isoelectric points were identified. Protein extracts of one of these OPN spots normalized the endometrial EGF concentrations on Day 3 in 41.9% of repeat breeder cows. The present results indicated that OPN may be the molecule responsible for the activity normalizing the EGF concentrations on Day 3 in the endometrium of repeat breeder cows

Effects of semen preservation procedure in egg yolk-tris based extender on bull spermatozoa characteristics

To verify the dynamics of damages to spermatozoa during semen freezing, characteristics of spermatozoa collected from 3 Japanese black bulls were evaluated by using fluorescent staining. Pre-diluted sample showed the highest proportion of spermatozoa with intact plasma membrane, intact acrosome and high mitochondrial potential. The proportion of spermatozoa with intact plasma membrane, intact acrosome, and low mitochondrial membrane potential were higher after cooling to 4°C than the other processes. During cooling preservation examined in this study, the proportion of spermatozoa with damaged acrosome increased. These results lead us to speculate that, during cooling process, spermatozoa may be firstly injured to mitochondrial membrane, and low mitochondrial function may cause the impairment of plasma membrane and subsequent cell death with acrosomal damage

Immunohistochemical localization of the estrogen receptor alpha (ERα) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) during pregnancy

Information on steroid hormone receptor distribution in the uterus is essential to understand the roles of their ligands in pregnancy. This study examined the spatio-temporal localization of estrogen receptor alpha (ERα) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) to determine the estrogen and progesterone action site during pregnancy. Ovaries and uteri were collected from 21 pregnant sika deer with single fetus and two corpora lutea, ranging from Day 20 to Day 207 of pregnancy. In addition, genital organs were also collected from three sika deer whose gestational status was unknown: one female had only one developing corpus luteum: ≤ Day 4 (metestrus) and two females had two corpora lutea, one of which was at the developing stage equivalent to diestrus or early pregnancy: > Day 7 (diestrus). Staining of ERα and PR was clear in all cell types during metestrus. During diestrus, the presence of ERα was also clear in deep glandular epithelium, stroma and myometrium, whereas it was suppressed in luminal epithelium and shallow glandular epithelium. Staining of PR was suppressed in luminal epithelium but was detectable in other cell types. Staining of ERα in all cell types and PR in luminal epithelium and glandular epithelium became undetectable by Day 28. PR was presented in stroma and myometrium throughout pregnancy. The distribution pattern of ERα and PR was different during diestrus from that in a ruminant. This could be attributed to estrogen secretion from the maturing and ovulating follicles in the presence of developed corpus luteum