論文の公開元へRole of metabolic and endocrine factors in an alteration of the endometrial epidermal growth factor concentration in repeat breeder dairy cows
概要
The performance of dairy herds depends on genetic selection for milk production and herd management. Milk production of dairy cattle has been increasing from 5,000 kg per year to 9,000 kg per year, from the mid-nineteenth century until the present day [1]. The percentage of animals that display standing estrus declined 80% to 50% and the first service pregnancy rate from 70% to 40%, suggesting that fertility has declined over the last 50 years while milk production is increasing [2,3]. In contrast, dairy cows have become delicate and fragile animals that require high levels of nutritional and cow comfort management to realize their genetic potential [4-6]. These changes inevitably result in the reduction of the fertility rate in modern dairy herds with the management of an average level.
Repeat breeder cows are defined as the cows which failed to conceive after several (usually three or more) inseminations without anatomical and infectious abnormalities, and with an apparently normal estrous cycle [7-11]. The incidence of repeat breeding in dairy cattle has been reported between 10% and 24% [10,12]. Repeat breeding syndrome has been a major cause of an economic loss of the dairy [13,14] and beef industries [15]. The causes of infertility in repeat breeding syndrome are unclear but include environmental, management, and animal factors [13]. Fertilization failure and embryonic death are thought to be major causes of repeat breeding, but mechanisms are not clearly understood [15]. The incidence of early embryonic loss increased during the last 40 years while fertilization rates were kept at a high level (about 90%) [16]. The incidence of high-quality embryos at 6-7 days after artificial insemination (AI) decreases in lactating cows (between 33% to 53%) compared with heifers (72%) and dry cows (83%) [17]. Together the retarded embryonic development might be common in high-yielding cows and become a major cause of infertility in repeat breeder cows [18]. Interestingly, a reciprocal embryo transfer study that exchanged embryos between fertile and repeat breeder cows showed that uterine environment, but not the quality of embryos, is the factor increasing embryonic loss in repeat breeder cows [19].
An increased level of milk production has a negative impact on the uterine environment and function due to decreased progesterone (P4) and estradiol-17β (E2) concentrations in the circulation [20]. The high-yielding cows need to intake a large amount of food to meet the high energy requirement, and this leads to increase liver blood flow [21] that, in turn, to increase the clearance of both P4 and E2 from the circulation [6]. Repeat breeder cows may not necessarily be high producers but exhibit similar alterations in the profiles of ovarian steroid hormones to those found in high producing cows [22].
The alterations of ovarian steroid hormones in high-yielding and repeat breeder cows could be amplified and become detectable in the endometrium as an alteration of growth factor and cytokine expression since ovarian steroid hormones regulate the expression of these local factors [22,23]. Epidermal growth factor (EGF) is one of the most important regulatory components of uterine function and embryonic development [23,24]. Estrogen stimulates EGF production in the uterus [25-27]. EGF replaces estrogen in the uterine, vaginal growth and lactoferrin (estrogen-inducible secretory protein) [28], and a nidatory estrogen surge that initiates blastocyst attachment to the endometrium in rodents [29,30]. Drastic negative effects on the number of inner cell mass [31], placenta formation, and viability of offspring [31,32] have been reported in mice.
The presence of EGF and its receptor in the endometrium have been reported in many farm animals, including cattle [13,33,34], sheep [35,36], goats [37], and pigs [38]. Moreover, EGF increases the production of prostaglandin (PG) E2 in the endometrium and the PGE2:PGF2α ratio in pigs [39] and rats [40]. These effects of EGF on PG synthesis enhance corpus luteum function and support the survival of embryo in cattle [41,42]. Therefore, an alteration of EGF action in the endometrium may cause uterine dysfunction and increase early embryonic loss in cattle.
The endometrial EGF exhibits the peak twice on day 2 to 4 and day 13 to 14 with lesser EGF concentrations around day 7 of the estrous cycle in fertile cows [13,43,44]. Loss of the two peaks in the EGF profiles has been linked to reduced fertility in repeat breeder and high-yielding cows [13,15,43,44]. This alteration of EGF concentrations was found in about 70% of lactating dairy repeat breeder cows and 40% of high-yielding cows at 60 days postpartum (dpp) [22,43]. However, the etiology of the abnormality of the EGF profile in the endometrium is not well understood. It is of interest to examine the process of recovery of EGF cyclicity in the endometrium during the postpartum period.
In chapter 1, I aimed to examine the recovery of endometrial EGF cycle in lactating cows, in which the regular estrous cycle resumed. Firstly, EGF profiles of normal cyclic dairy cows were examined in every estrous cycle from calving to 90 dpp to determine the changes of EGF cycle. Then, parity, body condition score (BCS) at calving, day of the first ovulation, peak milk yield, and time of the peak were considered as factors that may affect the recovery of endometrial EGF cyclicity. The study confirmed a potential association of high levels of milk production with delay of the recovery in the endometrial EGF cyclic change in lactating dairy cows.
Results of the chapter 1 prompted me to examine the role of the leptin system in the etiology of abnormality in the EGF profile in dairy cows. Leptin is a peptide hormone, a product of the obese (ob) gene [45,46]. Leptin expression is influenced by energy storage status and correlated to the degree of body fat mass in humans, rodents, and cattle [47-49]. More importantly, leptin plays an important role in the regulation of feed intake, energy expenditure, and endocrine function [50]. The leptin synthesis can be increased by glucocorticoids [51], lipid, and estrogen [52]. In contrast, testosterone [53], thyroid hormone [54], and β-adrenergic agonists [55] inhibit leptin synthesis. Exogenous leptin injection is found to restore fertility in infertile ob/ob mice [56]. Changes in leptin levels and mRNA expression in the adipose tissue were also associated with onset of puberty in cattle [57]. Leptin has been shown to control reproductive function via regulation of gonadotropin-releasing hormone (GnRH) secreting neurons [58], a key player in the hypothalamus-pituitary-gonadal axis. Leptin levels correlated with LH pulse frequency and amplitude in primiparous cows [59]. Intracerebroventricular administration of leptin induces hypersecretion of LH in fasted cows but not in well-fed cows[60].
Leptin receptor (Ob-R) is a member of class 1 cytokine receptor family [61] and express in six isoforms consisting of a full-length isoform (Ob-Rb), short isoforms (Ob-Ra, Ob-Rc, ObRd, Ob-Rf) and soluble isoform (Ob-Re) [62]. Expression of Ob-Rb, Ob-Ra and Ob-Rc were reported in peripheral tissues of cattle [63-65]. Expression of Ob-R transcript is the second most abundant in the uterus next to the liver among the peripheral tissues of prepubertal dairy heifers [64]. Among the various isoforms, the sum of Ob-Ra and Ob-Rb transcripts accounted for nearly the total amount of Ob-R, and Ob-Ra accounted for most of the Ob-R in the bovine uterus [64]. Estrogen suppresses Ob-R expression in prepubertal dairy heifers [64]. Ob-R levels in cyclic heifers were high during the luteal phase and the lowest on day 5 of the estrous cycle, with intermediate levels at estrus [66]. The lowest expression of Ob-R coincides with the first peak of endometrial EGF concentrations [13,44]. Together with the regulatory role of estrogen in endometrial EGF concentrations, Ob-R may be involved in the etiology of the abnormality in the endometrial EGF profile.
I had a particular interest in the role of the leptin system to determine the response of repeat breeder cows to the therapeutic treatment either with progesterone and estradiol [67] or seminal plasma (SP) [68]. Both treatments normalized the EGF profile in about 60% of repeat breeder cows [69] but factors segregating the consequence of treatment are unknown. Currently, this is a major obstacle to make the measures more effective. However, there is a preliminary observation that may be a clue to solve this problem. Repeat breeder beef cows with excessively high BCS and high leptin levels are resistant to treatment, and reduction of BCS by feed restriction and forced exercise reduced leptin levels and restored response to treatment [69]. It is likely that alteration of the leptin system may prevent repeat breeder cows from responding to treatment. Alternatively, I could find some difference in the leptin system in repeat breeder cows that were responding or not responding to treatment.
In chapter 2, I hypothesized that the leptin system may have a role in determining the response of therapeutic treatments to normalize the EGF profile in repeat breeder cows. The leptin system, the plasma leptin concentration and Ob-R expression in the endometrium, were examined in heifers, normal cows (fertile control), and repeat breeder cows together with EGF concentrations, before and after SP treatment to normalize the EGF profile. The data indicated that endometrial Ob-R, but not circulating leptin levels, may be related to the abnormality of the EGF profile in the endometrium.
