Nhlh2 is a member of the basic helix-loop-helix (bHLH) transcription factor family and is expressed in developing and adult neuroendocrine tissues such as the pituitary and hypothalamus. Targeted deletion of Nhlh2 (N2KO) in mice results in hypogonadism and obesity. While gonadally intact male N2KO mice are infertile and lack male sexual behavior, female N2KO mice can become pregnant and carry litters to full term. Unlike normal females in which fertility averages 8–12 months with approximately one pregnancy per month, N2KO females have a shorter reproductive span with most females supporting only three to four pregnancies in a 9-month period. In addition, N2KO females exhibit abnormal estrous cycles characterized by a truncated estrus and a prolonged proestrus. We have found that while young female N2KO mice ovulate the same number of oocytes as normal females in response to exogenous hormones, the number of oocytes released by aged N2KO females is reduced over 50%. Interestingly, oocytes from N2KO females are equally competent for in vitro fertilization assays when compared to oocytes from similarly aged normal and heterozygous mice. We have further demonstrated that both young and old N2KO females show at least a 50% reduction in hormone-stimulated sexual behavior as measured by their lordosis quotient. This suggests that N2KO females show a lifelong behavioral hyporesponsiveness to exogenous steroid hormones accompanied by a reduction in reproductive longevity via reduced ovulation with aging. Potential gene regulatory mechanisms that involve the action of the Nhlh2 transcription factor on female fertility and sexual behavior are discussed.
At a minimum, successful reproduction requires that a female produce functional oocytes and demonstrate mating behavior in response to the male. If either of these are lacking, fertility of the animal will be reduced. It is well established that estrogen (E) and progesterone (P) are required for both sexual behavior and ovulation Etgen et al., 1999 and Pfaff et al., 2002. In addition, gonadotropin hormone releasing hormone (GnRH) must be released into the hypophyseal portal to trigger production and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH) into the circulation. In female mammals, these hormones are part of a regulatory loop in which FSH stimulates follicular growth as well as production of E, which feeds back to the hypothalamus, providing a positive signal that results in the LH surge. This, in turn, results in ovulation and production of P by the corpus luteum. In the mouse, the entire estrous cycle takes approximately 5 days with animals spending the most time in the diestrus and estrus phases of the cycle (Rugh, 1990).
In normal, intact females, sexual behavior is elicited during the estrus phase of the cycle when P levels are high, which follows high E levels in the late pro- and early estrus phases. During estrus, introduction of a male leads to a hormonally dependent, reflexive display of copulatory behavior called lordosis, which is characterized by an arching of the back and a rigid stance that facilitates male copulation. Ovariectomy obliterates lordosis by removing the sources of E and P, but treatment of gonadectomized mice sequentially with E and P restores this receptive behavior. Receptors for E and P are produced in the hypothalamus and medial preoptic area (MPOA) and an extensive amount of research supports their role in genomic regulation of female reproductive behavior and fertility. For example, mice with a deletion of the alpha form of the estrogen receptor (ERαKO) display no lordosis response when either gonadally intact (Ogawa et al., 1996) or when ovariectomized and administered steroids Ogawa et al., 1998 and Rissman et al., 1997. Furthermore, when stud males are placed in a cage with intact ERαKO females in diestrus, these females are attacked more often and mounted less than normal females (Ogawa et al., 1996). In contrast, females with a deletion of the beta form of the estrogen receptor show normal sexual receptivity (Ogawa et al., 1999). Deletion of both the A and B forms of the progesterone receptor in mice (PRKO mice) ameliorates female sexual behaviors Lydon et al., 1995 and Lydon et al., 1996.
In addition to the MPOA, neurons in the ventromedial hypothalamus (VMH) are necessary for female sexual behavior and reproduction. Rodents with VMH lesions are not receptive to males (Pfaff and Sakuma, 1979) while electrical stimulation of the VMH can result in lordosis behavior with exogenous hormone treatment. Furthermore, implantation of fetal preoptic area explants into the third ventricle, adjacent to the VMH, results in resumption of lordosis in mice carrying a mutation of the GnRH gene (Gibson et al., 1987), as does injection of estrogens into this area in ovariectomized rats (Butera and Beikirch, 1989). Receptors for both E and P are present in the VMH Garris et al., 1983 and Koch and Ehret, 1989.
