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a Instituto de Investigaciones Biomédicas, Universidad Nacional Autonoma de Mexico, 04510 México, D.F., Mexico
b Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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With one exception [2] it was found that YTIR.B6 (previously named YDOM.B6) females are sterile. Histological examination revealed that their ovaries (designated as XYTIR ovaries hereafter) had a smaller number of follicles than XX ovaries; thus there was a depletion of oocytes between 4 and 8 wk of age [2, 4]. A study of XYTIR fetal ovaries showed that this was due to a massive death of oocytes at 16–18 days postcoitum, mainly in the medullary region [6]. One to three weeks after birth, the medullary cords devoid of oocytes formed a luteinized-like tissue that produced abnormal levels of steroid hormones [7]. After birth, the remaining oocytes in XYTIR ovaries formed morphologically normal follicles. Furthermore, after treatment with gonadotropins, XYTIR.B6 females mated and ovulated, and their oocytes were fertilized. Some fertilized eggs started cleavage but failed to implant [8].
Fertilized eggs from XYTIR ovaries may be unable to develop owing to an inhibitory factor kept in XYTIR oocytes produced by the YTIR chromosome during oogenesis and/or owing to sex chromosome univalence [8, 9]. Alternatively, the abnormal XYTIR oocyte may disturb differentiation of cumulus and granulosa cells, which may in turn impair follicle development. Since there is an abnormal production of steroid hormones [7] and irregularities of the estrous cycle [6], the chance of reproduction of YTIR.B6 sex-reversal females may be reduced because of an alteration of the hypothalamus-pituitary-ovary axis (HPOa). Some observations suggest that at least part of the HPOa remains functional. When XYTIR ovaries were placed under the kidney capsule (heterotopic transplant) of ovariectomized XX females, their estrous cycles were interrupted. However, when XX ovaries were heterotopically transplanted into YTIR.B6 females, several mice exhibited regular estrous cycles [6]. Nevertheless, the capacities of the YTIR.B6 sex-reversal females to maintain pregnancy, deliver offspring, and feed their pups are other reproductive functions of the HPOa that may have been affected by the impaired ovarian development and/or the presence of the YTIR chromosome in the hypothalamus and pituitary.
It was reported that a significant number of ovariectomized mice recovered estrous cycles and that several became fertile when fetal [10, 11] or postnatal [12–14] ovaries were transplanted under the ovarian capsule (orthotopic transplant). In the present study we used this technique to obtain insights into the following questions: 1) Do morphologic and endocrine alterations observed in fetal and juvenile XYTIR ovaries cause a permanent alteration in the HPOa of YTIR.B6 adult sex-reversal females? 2) Do XYTIR ovaries become fertile when placed onto B6 normal females?
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research of the National Academy of Sciences, Bethesda, MD (1996). YTIR.B6 mice kept in our mouse colony (backcross generations N19–23) that possess the B6 genetic background and the Y chromosome from M. musculus domesticus (from Tirano, Italy) were produced as previously described [4, 6]. The YTIR.B6 progeny were produced by crossing YTIR.B6 males with B6 females (a kind donation from Dr. Teruko Taketo, McGill University). The sexual genotype of each YTIR.B6 phenotypic female was determined by dot hybridization of peripheral blood with the previously described DNA probe 145SC5 specific for mouse Y chromosome [15].
Ovarian Graft Donors
Each YTIR.B6 or B6 fertile male (50 days postpartum, dpp) was placed with three B6 females (50–60 dpp). Females were examined for the presence of a vaginal plug every morning between 0900 and 1100 h. The day the vaginal plug was found was defined as Day 0 postcoitum. The day of birth was defined as Day 0 postpartum. For graft experiments, ovaries from both B6 and YTIR.B6 phenotypic females were taken at 17–19 days postcoitum (fetal), at 2–15 dpp (infantile), and at 5–11 wk (adult).
Preparation of Ovarian Grafts
Postnatal B6 and YTIR.B6 sex-reversal female donors were killed by cervical dislocation, and their ovaries were dissected, liberating them from the ovarian capsule, and maintained in a Petri dish with L-15 medium (Leibovitz's-15 medium; Gibco BRL, Gaithersburg, MD) complemented with 200 UI/ml of G sodic penicillin and 200 µg/ml of streptomycin sulfate until transplanted. To obtain fetal ovaries, pregnant females were killed by cervical dislocation and fetuses were extracted from the uterine horns. After decapitation, the fetal abdomen was opened; the ovaries were dissected from the ovarian capsule and maintained in a Petri dish with L-15 medium complemented with 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO), 200 UI/ml of G sodic penicillin, and 200 µg/ml of streptomycin sulfate until transplantation.
Orthotopic Transplantation
Recipient females were anesthetized with an i.p. injection of chloral hydrate (3.5% solution of chloral hydrate) in a dose of 1 ml/100 g BW. The orthotopic transplantation procedure was a modified version of the method of Robertson [16]. Female recipients were ovariectomized either unilaterally or bilaterally. Through an incision directly on the ovarian capsule along the fat pad, each ovary was removed at the hilum. One donor ovary was then placed into each of the empty ovarian capsules, and the incision was sewn up using polypropylene thread (Prolene 7-0; Ethicon, Mexico City, Mexico). Reproductive organs were returned into the abdominal cavity, and its wall was then sutured with polypropylene thread. The skin was finally sutured with silk thread 3-0 (Ethicon). The whole procedure was performed under a stereomicroscope (Nikon SMZ-2T; Garden City, NY). Ovarian transplants of B6 or XYTIR at different ages were grafted either unilaterally or bilaterally as shown in Table 1.
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Assessment of Fertility of Recipient Females
After 15–20 days of postsurgical recovery [17], females were caged with fertile B6 males and monitored for the presence of vaginal plugs. Pregnant females were isolated and observed in order to determine whether gestation came to term. After delivery, the number of pups produced per litter was registered, as well as the numbers of pups that died and that completed their lactation period. Females were killed by cervical dislocation at different times: 1) after all their pups had died soon after birth, 2) after the conclusion of the period of lactation, or 3) 9–12 wk after operation if they had not become pregnant. Control, bilaterally ovariectomized adult mice were monitored for the presence of vaginal plugs, estrous cycles, and pregnancy.
Histological Study of Grafted Ovaries
Ovaries were fixed in Karnovsky solution [18] without Ca2+ (pH 7.4), postfixed in 1% OsO4 in Zetterqvist buffer [19], and embedded in Epon 812 (Electron Microscopy Sciences, Ft. Washington, PA). Semithin sections (1 µm) were stained with toluidine blue.
Statistical Analysis
Data are presented as the percentage of those females carrying ovarian grafts that became pregnant. Significant differences were determined using chi-square analysis (P > 0.01). Significant differences between the means at a particular age of the grafted ovary in B6 or XYTIR.B6 female hosts were determined by t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results are summarized in Table 2 and Figure 1; 8 B6 and 7 YTIR.B6 females with fetal XX ovaries, from a total of 21 recipient females in each group, became pregnant (38% and 33%, respectively). Pregnancy occurred within 5–7 wk after transplantation. B6 host females with transplanted fetal ovaries had 4.3 ± 1.4 pups per litter, and YTIR.B6 female hosts had 4.4 ± 2.5. No significant difference was found between the two groups (Fig. 1). The age of ovarian grafts at the time when host females became pregnant was 47.0 ± 16.4 dpp. Pregnancy occurred in females with either bilateral or unilateral transplant.
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Of 15 B6 females with infantile XX transplanted ovaries, 66% became pregnant, whereas of 16 XYTIR.B6 recipient females, 75% were pregnant (Table 2). The mice took from 4 to 5 wk after transplantation to become pregnant. Although the percentage of female hosts carrying infantile ovaries was similar between the two groups, significant differences were found in the number of pups per litter. B6 females had 3.2 ± 1.8 pups per litter whereas YTIR.B6 females had 6.7 ± 3.6 (Fig. 1). The age of the ovarian grafts at the time of pregnancy was 43.0 ± 11.4 dpp. Pregnancy occurred both in females with bilateral and in those with unilateral infantile ovarian transplants.
With transplants of adult ovaries, 14 of 15 recipient B6 females became pregnant (93%) and 9 of 15 YTIR.B6 female hosts became pregnant (60%) within 3–6 wk after transplantation (Table 2). The number of pups per litter was 4.9 ± 1.4 and 6.2 ± 2.6 for B6 and XYTIR.B6, respectively (Fig. 1). The chronological age of the grafted ovaries at the time of pregnancy was 79.6 ± 16.3 dpp. Both females with bilateral and those with unilateral adult ovarian transplants became pregnant. Most female hosts in the two groups carrying fetal, infantile, or adult grafts started estrous cycles after 2 or 3 wk of transplant. After delivery, no differences were observed between B6 and XYTIR.B6 female hosts in feeding and taking care of their pups.
B6 Females with Orthotopic XYTIR Ovarian Transplants
None of the 15 B6 female recipients carrying fetal, infantile, or adult XYTIR transplanted ovaries became pregnant. Autopsy showed that all animals had retained grafts either in one or in both ovarian capsules. Oviducts and uterus were placed normally, and formation of adhesions was absent in most recipient females. Estrous cycles were detected after 2 or 3 wk of operation. All females carrying infantile XYTIR ovaries showed at least two cycles, whereas 20% of female hosts with fetal or adult XYTIR ovaries failed to show vaginal cycles.
Histology of Transplanted Ovaries
Fetal, infantile, and adult XX ovaries transplanted to XYTIR.B6 or B6 females presented histological characteristics similar to those of normal adult ovaries. Primordial, preantral, graafian, and atretic follicles were evident. Abundant interstitial cells were present in the stromal tissue. Several healthy corpora lutea and some undergoing resorption were also present. Although detection of innervation was not attempted, revascularization of the grafted ovaries was clearly observed (Fig. 2, A and B). All XYTIR ovaries transplanted to B6 female hosts showed complete absence of follicles 9–12 wk after operation. The ovaries appeared to be filled either with fibrotic tissue or with irregular clusters of luteal-like cells of different sizes. Within some clusters, vestigial zona pellucida suggested that these were corpora lutea atretica where ovulation was prevented (Fig. 2, C and D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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The lower percentage of pregnancy obtained with adult ovaries in XYTIR.B6 females as compared to B6.XX female hosts can be explained by our increased experience in the surgical procedure during the study. In first operations, adult B6 ovaries were transplanted to XYTIR.B6 female hosts, and adult XYTIR ovaries were transplanted to B6 female hosts. When we found that none of the B6 females became pregnant with adult XYTIR ovaries, B6 female hosts carrying adult XX ovaries were used as controls in further experiments. Although significant differences were observed in the number of pups per litter with infantile ovaries, no significant difference was found between the two groups when ovarian grafts at fetal and adult stages were added.
It is difficult to discern the factors that alter fertility in ovarian grafts. Most cases of infertility were due to alterations caused by surgical stress and the postoperative sequelae that precluded implantation. Often, females with ovarian implants showed distension of the oviduct and/or the ovarian capsule caused by a clear fluid that may result in impaired fertilization or egg displacement. The low pregnancy rates seen in females with fetal ovarian transplants may have been primarily due to a higher loss of oocytes in fetal than in postnatal ovaries during the postoperative period [13]. Some fetal ovaries were possibly not functional due to lack of adequate innervation and/or vascularization [20].
Considering that in adult mice, it takes approximately 22 days in ovaries for a primordial follicle to reach the stage of ovulation [21], fetal XX ovaries showed accelerated maturation after orthotopic transplantation into XYTIR.B6 females. Precocious maturation of transplanted fetal ovaries has been observed previously [10, 11]. Cox et al. [11] suggested that the rise in concentration of circulating gonadotropins following ovariectomy may provide the stimulus for the accelerated development of fetal ovaries. They found that in unilaterally ovariectomized female mice, the transplanted fetal ovary was unable to recover functionality. An inhibitory effect from the host ovary over the ovarian transplant was suggested [11]. In the present study, unilaterally and bilaterally ovariectomized XYTIR.B6 females with XX ovarian transplants had similar reproductive performance, suggesting that when left, the host XYTIR ovary did not produce the putative inhibitory factor.
Previous studies showed that although XYTIR.B6 females ovulate when treated with gonadotropins, the span of the ovulatory response was shorter than in B6 females. The small number of oocytes left after birth in XYTIR ovaries may be responsible for the impaired ovulation [9]. In the present study, none of the B6 females with XYTIR ovarian transplant became pregnant, although they did show a vaginal plug indicating that they had mated. Histologic analysis showed that XYTIR transplanted ovaries lacked follicles after 9–12 wk. Two aspects of the transplanted XYTIR ovaries may cause their infertility: the high loss of oocytes seen in fetal XX ovaries is aggravated in XYTIR ovaries already depleted of oocytes [9]. Postnatal XYTIR ovaries, on the other hand, have fewer follicles than XX ovaries to be rescued after transplantation.
The present results demonstrate that XYTIR.B6 sex-reversal females became fertile with transplanted XX ovaries and that after delivery they showed normal maternal behavior in feeding and caring for their pups. The current study suggests that XYTIR.B6 sex-reversal females maintain a functional HPOa and confirms that the presence of the YTIR chromosome affects fertility only at the ovarian level [22].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: H. Merchant-Larios, Department of Cell Biology, Instituto de Investigaciones Biomedicas, UNAM, Apartado Postal 70228, Ciudad Universitaria, Mexico, D.F. Mexico 04510. FAX: 525 622 3897; merchant{at}servidor.unam.mx
Accepted: July 15, 1999.
Received: March 23, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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  N. Moreno-Mendoza, L. Torres-Maldonado, J. Chimal-Monroy, V. Harley, and H. Merchant-Larios Disturbed Expression of Sox9 in Pre-Sertoli Cells Underlies Sex-Reversal in Mice B6.Ytir Biol Reprod, January 1, 2004; 70(1): 114 - 122. [Abstract] [Full Text] [PDF]
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