Maria E. Ayala

Oogenesis in adult mammals, including humans: a review.

The origin of oocytes and primary follicles in ovaries of adult mammalian females has been a matter of dispute for over 100 yr. The prevailing belief that all oocytes in adult mammalian females must persist from the fetal period of life seems to be a uniquely retrogressive reproductive mechanism requiring humans to preserve their gametes from the fetal period for several decades. The utilization of modern techniques during last 10 yr clearly demonstrates that mammalian primordial germ cells originate from somatic cell precursors. This indicates that if somatic cells are precursors of germ cells, then somatic mutations can be passed on to progeny. Mitotically active germline stem cells have been described earlier in ovaries of adult prosimian primates and recently have been reported to also be present in the ovaries of adult mice. We have earlier shown that in adult human females, mesenchymal cells in the ovarian tunica albuginea undergo a mesenchymal-epithelial transition into ovarian surface epithelium cells, which differentiate sequentially into primitive granulosa and germ cells. Recently, we have reported that these structures assemble in the deeper ovarian cortex and form new follicles to replace earlier primary follicles undergoing atresia (follicular renewal). Our current observations also indicate that follicular renewal exists in rat ovaries, and human oocytes can differentiate from ovarian surface epithelium in fetal ovaries in vivo and from adult ovaries in vitro. These reports challenge the established dogma regarding the fetal origin of eggs and primary follicles in adult mammalian ovaries. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. Yet, the follicular renewal may cease at a certain age, and this may predetermine the onset of the natural menopause or premature ovarian failure. A lack of follicular renewal in aging ovaries may cause an accumulation of spontaneously arising or environmentally induced genetic alterations of oocytes, and that may be why aging females have a much higher chance of having oocytes with more mutations in persisting primary follicles.

Changes of ovarian interstitial cell hormone receptors and behavior of resident mesenchymal cells in developing and adult rats with steroid-induced sterility.

In the present paper, we report that injection of testosterone propionate (500 microg) during the critical window of rat development (postnatal day 5) induces temporary appearance of aged interstitial cells in developing ovaries (days 7 and 10). Aged interstitial cells showed large size (> or = 12 microm), enhanced androgen receptor (AR) and low estrogen (ER) and luteinizing hormone receptor (LHR) expression. Although normal mature interstitial cells (large size and strong ER and LHR expression) appeared later (day 14), and ovaries of androgenized rats were similar to normal ovaries between days 14 and 35, ovaries of adult androgenized females showed only aged and no mature interstitial cells. Androgenization on day 10 caused the development of aged interstitial cells on day 14, but adult ovaries were normal. Long lasting postnatal estrogenization (estradiol dipropionate for four postnatal weeks) caused in developing and adult ovaries a lack of interstitial cell development beyond the immature state. Immature interstitial cells were characterized by a small size (< or = 7 microm) and a lack of AR, ER and LHR expression. Because the critical window for steroid-induced sterility coincides with the termination of immune adaptation, we also investigated distribution of mesenchymal cells (Thy-1 mast cells and pericytes, ED1 monocyte-derived cells, CD8 T cells, and cells expressing OX-62 of dendritic cells) in developing and adult ovaries. Developing ovaries of normal, androgenized and estrogenized females were populated by similar mesenchymal cells, regardless of differences in the state of differentiation of interstitial cells. However, mesenchymal cells in adult ovaries showed distinct behavior. In normal adult ovaries, differentiation of mature interstitial cells was accompanied by differentiation of mesenchymal cells. Aged interstitial cells in ovaries of androgenized rats showed precipitous degeneration of resident mesenchymal cells. Immature interstitial cells in ovaries of estrogenized rats showed a lack of differentiation of resident mesenchymal cells. These observations indicate that an alteration of interstitial cell differentiation during immune adaptation toward the aged phenotype results in precipitous degeneration of resident mesenchymal cells and premature aging of ovaries in adult rats, and alteration toward immature phenotype results in a lack of differentiation of mesenchymal cells and permanent immaturity of ovaries in adult females.

Bone Marrow Derived Cells and Alternative Pathways of Oogenesis in Adult Rodents

Not yet available.

Study Origin of Germ Cells and Formation of New Primary Follicles in Adult Human and Rat Ovaries

The central thesis regarding the human ovaries is that, although primordial germ cells in embryonal ovaries are of extraovarian origin, those generated during the fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE) bipotent cells. With the assistance of immune system-related cells, secondary germ cells and primitive granulosa cells originate from OSE stem cells in the fetal and adult human gonads. Fetal primary follicles are formed during the second trimester of intrauterine life, prior to the end of immune adaptation, possibly to be recognized as self-structures and renewed later. With the onset of menarche, a periodical oocyte and follicular renewal emerges to replace aging primary follicles and ensure that fresh eggs for healthy babies are always available during the prime reproductive period. The periodical follicular renewal ceases between 35 and 40 yr of age, and the remaining primary follicles are utilized during the premenopausal period until exhausted. However, the persisting oocytes accumulate genetic alterations and may become unsuitable for ovulation and fertilization. The human OSE stem cells preserve the character of embryonic stem cells, and they may produce distinct cell types, including new eggs in vitro, particularly when derived from patients with premature ovarian failure or aging and postmenopausal ovaries. Our observations also indicate that there are substantial differences in follicular renewal between adult human and rat ovaries. As part of this chapter, we present in detail protocols utilized to analyze oogenesis in humans and to study interspecies differences when compared to the ovaries of rat females.

Effects of a lesion in the dorsal raphe nuclei performed during the juvenile period of the female rat, on puberty

The present study examined the effect of thermic lesions on the dorsal raphe nuclei (DRN) of the female rat, performed at various ages during the prepubertal period (21, 24, or 27 days), on puberty, and at first ovulation. In comparison with sham-operated animals, the age of vaginal opening and first vaginal oestrus was delayed in rats with a DRN lesion performed at the end of the infantile period (day 21) (45.6+/-0.94 vs. 89.9+/-1.03, p < 0.05), whereas differences were not observed in animals with lesions performed during the juvenile period (day 24 or 27). The number of ova shed by ovulating animals was greater in those rats with lesions performed on day 24 or 27 (9.7+/-0.4 vs. 7.4+/-0.4; 9.5+/-0.5 vs. 7.7+/-0.4, p < 0.05). Ovarian follicular atresia in these animals was significantly lower than in control and sham-operated ones. On the day of first vaginal oestrus and 48 h after the lesion, the serotonin-hypothalamic concentration decreased in all lesioned animals. Present results support the idea of the participation of the serotoninergic system, arising from the DRN, in the neuroendocrine mechanisms regulating the onset of puberty and the first ovulation, with variations depending on animal maturity.

Different effects of unilateral and bilateral lesions of the dorsal raphe nucleus on puberty and first ovulation

The effects of unilateral and bilateral electrolytic lesions of the dorsal raphe nuclei (DRN) of 30-day-old rats, on the spontaneous and induced ovulation, were analyzed. The bilateral lesion and the lesion on the right side of the DRN delayed the age of first vaginal estrous. None of the animals with bilateral lesion on the DRN ovulated on the day of first estrous (0/8 vs. 12/15, p < 0.05). The ovulation rate in animals with unilateral lesion on the DRN was similar to sham-operated animals, but the number of ova shed by ovulating animals increased in the ovary ipsilateral to the lesion (lesion on right DRN, right ovary: 6.5 +/- 0.5 vs. 4.5 +/- 0.4; lesion on left DRN, left ovary: 6.4 +/- 0.3 vs. 4.2 +/- 0.4, p < 0.05). By the injection of human chorionic gonadotropin, ovulation was restored in rats with a bilateral lesion on the DRN (3/5 vs. 0/8, p < 0.05). The present results suggest that serotoninergic input to the hypothalamus, arising from the DRN, exerts a facilitatory influences on the control of luteinizing hormone release. To explain the increase in the number of ova shed by the left and right ovary, observed in rats with an ipsilateral lesion, we suggest the existence of a neural connection between the DRN and the ovary.

Comparative effects of injecting 5,6-dihydroxytryptamine in the dorsal or medial raphe nuclei on rat puberty

The role played by the serotoninergic system in the control of puberty onset and first ovulation in rats is studied in this paper by analyzing the effects of injecting the neurotoxin 5,6-dihydroxytryptamine (5,6-DHT) into the dorsal (DRN) or medial (MRN) raphe nucleus of 30-day-old female rats. Complete lesion to the DRN resulted in the blockade of ovulation and a decrease in both the number of ovarian follicles and the serum concentration of follicle stimulating hormone (FSH). This treatment was also found to be associated with an increase in serotoninergic activity in the anterior and medial hypothalami. A lesion to the central portion of the DRN resulted in a significant decrease in the concentration of progesterone in serum and in the number of ova shed by ovulating animals. The lesion to the lateral portion of the DRN did not have an apparent effect on ovulation rate, the number of ova shed, nor in hormone serum concentration. The injection of propranolol to rats with a lesion to the DRN restored ovulation in 73% of treated animals and returned serotoninergic activity in the anterior hypothalamus to levels similar to those of sham-operated animals. In turn, in the medial hypothalamus, the increase in serotoninergic activity was not modified. The results presented herein suggest that serotoninergic inputs to the anterior hypothalamus have a direct influence on gonadotropin secretion and first ovulation, while the noradrenergic innervation exerts an indirect influence.

Effects of systemic administration or intrabursal injection of serotonin on puberty, first ovulation and follicular development in rats

To elucidate the role of serotonin in the onset of puberty, the effects of both systemic and in-ovarian bursa administration of serotonin on the neuroendocrine mechanism that modulates the onset of puberty, follicular development and first ovulation were evaluated. Two experiments were carried out. For the first, 25 or 37.5 mg kg⁻¹ of bodyweight of serotonin creatinine sulfate was administered by a subcutaneous route to 30-day-old female rats. In the second experiment, serotonin creatinine sulfate was administered directly into the ovarian bursa of 34-day-old female rats. Systemic administration of 25 or 37.5 mg kg⁻¹ of serotonin creatinine sulfate induced a delay in the ages of vaginal opening and first vaginal oestrus, a decrease in the number of ovulating animals, and serum concentrations of FSH, LH, oestradiol and progesterone. An increase in the number of Class 3 (>500 μm) and atretic follicles was observed in the ovaries of these animals. The administration of serotonin creatinine sulfate in the ovarian bursa did not modify the onset of puberty and ovulation, but a reduced serum concentration of oestradiol was observed. Our results suggest that serotonin acts on the components of the hypothalamus-hypophysis-ovary axis by modulating follicular development, ovarian functions and the onset of puberty.

Changes in monoaminergic activity in the anterior, medium and posterior hypothalamus, gonadotropins levels and ovarian hormones during puberty of the female rat.

The aim of present study is the analysis of monoamines concentrations changes in the anterior, medium and posterior hypothalamus, as well as changes in serum gonadotropins levels, ovarian steroids and follicular growth during the prepubertal development of the female rat. Noradrenergic activity in the anterior, medium and posterior hypothalamus reached highest level at day 13 after birth, followed by a subsequent decrease from day 15 to 19 and an increase on days 22 and 27 postnatal. At day 1, neural activity in the medium hypothalamus was higher than the activity in the anterior and posterior hypothalamus. Serotoninergic activity in three portions of the hypothalamus was higher throughout the prepubertal development. Follicle-stimulating hormone and luteinizing hormone serum levels increased between days 11 and 17 and decreased from day 19 to 36. The concentration of 17beta-estradiol was consistently low throughout the prepubertal development and increased at day 39 after birth. These results indicate that during the prepubertal development of the rat, the three regions of the hypothalamus show significant changes in the monoaminergic neural activity. There is an inverse relationship between the noradrenergic activity on the anterior and medium hypothalamus and serotoninergic activity in the posterior hypothalamus with ovarian steroids during sexual maturation. These changes may be linked to the development of the neuroendocrine processes that modulate gonadotropin secretion and ovarian function.

Impaired serotonin communication during juvenile development in rats diminishes adult sperm quality

ABSTRACT Spermatogenesis and steroidogenesis are testicular functions regulated by gonadotrophins as well as other factors, including serotonin. Testicular serotonin acts as an autocrine regulator of testosterone secretion, but studies on its role in spermatogenesis and sperm quality are scarce. Here, we analyzed the effects of intratesticular inhibition of serotonin synthesis on gonadotrophins, testosterone, and sperm quality. Both testicles of 30-day-old rats were injected once with saline solution (SS) or distinct concentrations of p-chloroamphetamine (PCA) (0.03, 0.06, or 0.12 mg). At 65 days of age, rats were euthanized and sperm density, motility, membrane integrity, mitochondrial function, and abnormalities were evaluated in gametes from the vas deferens. Inhibition of synthesis of intratesticular serotonin by PCA diminished the concentrations of testosterone and follicle-stimulating hormone (FSH) but luteinizing hormone (LH) levels were unaltered. Sperm density was not modified in animals injected with the different concentrations of PCA. In contrast, the percentage of sperm with abnormalities increased and the sperm membrane integrity decreased in animals injected at higher PCA concentrations. The functionality of sperm mitochondria in PCA-injected animals decreased only at the highest PCA dose. Our results indicate that testicular serotonin plays a role in testosterone synthesis and in the normal development of sperm, and blocking its effects disrupts the hormonal communication between the testis and hypophysis. Abbreviations: SS: saline solution; PCA: p-chloroamphetamine; FSH: follicle-stimulating hormone; LH: luteinizing hormone; TPH: tryptophan hydroxylase; MAO: monoamine oxidase; AC: absolute control group; PI: propidium iodide; FLICA: fluorescence inhibitor of caspase; 3β-HSD: 3β-hydroxysteroid dehydrogenase; 17-KSR: 17-ketosteroid reductase; DHT: 5-dihydrotestosterone