Attempts to control differentiation of the reproductive system of vertebrates have been made by use of hormone treatment, grafting, and parabiosis of embryos, larvae and neonates (for reviews, see Domm, 1939; Burns, 1949; Willier, 1952; Witschi, 1961; Burns, 1961). True sex reversal by use of steroid compounds has been accomplished only in certain amphibians (Witschi & Chang, 1950; Chang & Witschi, 1955) and in more primitive forms. Among birds and mammals such manipulations have modified components of the reproductive system but have not resulted in ovulation and fertilization of ova from the cortex of feminized gonads of the male. Spermatogenesis in the hypertrophied right rudiment of a small percentage of hens ovariotomized prior to 30 days has been reported by Domm (1929) but in only rare instances has it occurred in birds similarly treated at an older age (Domm, 1927). No chicks have hatched from eggs of hens artificially inseminated with sperm derived from such experimentally modified birds.

Pincus & Erickson (1962) described the results of dipping the fertile chick egg, between 0–5 days of incubation, into solutions of diethylstilbestrol. Differentiation of the male reproductive system was modified in a manner comparable to that seen following injection of estrogens (Domm, 1929; Willier et al. 1937). A cortex developed on the left gonad, the genital papilla was reduced to the form characteristic of the female, and portions of the Mullerian system were often retained. Apparently only Domm (1939) has reported persistence of some cortical material in adult male gonads following estrogen treatment limited to embryonic stages. Boss & Witschi (1947) reported persistence of the cortical portion of ovotestes which were induced by estrogen treatment of herring gull embryos on the 3rd, 6th or 9th day of incubation. Injections were continued beyond hatching and, as a result of this supportive treatment, cortical elements persisted up to 4 years in treated males. Others who have reported on the subsequent fate of feminized gonads of male chicks have described persistence of some portions of the medullary component but regression of the cortex (Wolff, 1935; vanTienhoven, 1957; Haye, 1959).

This paper reports on the results of one approach which has been made to the problem of maintaining the cortex of the feminized testis in the chick. Cortical and medullary development of ovotestes implanted in intact females was compared with cortical and medullary development of the host ovary and implants of normal testes and ovaries, to determine if isolation from an apparently unmodified right testis combined with residence in the female organism might present an endocrine environment more favorable to the persistence of the cortical elements. We were further interested in the fate of such early gonadal grafts—looking for an advantage similar to that described by Cannon & Longmire (1952) and Cannon (1954) who reported that the highest percentages of successful skin homografts to chicks were made immediately after hatching with diminishing success as implantation was delayed.

Merryknoll chicks were used in all experiments. These birds are a cross of Rhode Island Red males with Barred Plymouth Rock females to give a sex-finked white feather spot on the head of the male (which persists in spite of steroid treatments used to date) in contrast to the black head of the female. Ovotestes were obtained from newly hatched (0–3 days of age) males which developed from eggs that had been immersed at 96 hr. of incubation for 10 sec. in 40 mg. per cent, diethylstilbestrol dissolved in 100 per cent, isopropyl alcohol held at 10–12°C. Whole ovotestes, or longitudinal thirds to halves of ovaries or testes, were implanted. The hosts were 1–3 day sex-linked females. They were anesthetized with 0·03–0·04 ml. of Nembutal (60 mg./ml.) intraperitoneally. The feathers were plucked from the left side over the ribs and anterior margin of the thigh. An incision approximately 2 cm. long was made through the skin. Connective tissue between the body wall and the iliotibialis muscle was teased free and the last intercostal space located. A puncture, made with the tips of fine forceps, was spread to allow introduction of rib retractors. Under a dissecting microscope at 15 × the implant was placed beneath the peritoneum overlying the anterior margin of the left kidney, or in some cases under the thin fold of peritoneum which is reflected over the surface of the ovary. The opening between the ribs was closed with two to three sutures of 6–0 braided black silk and the skin approximated with a piece of adhesive tape. Mortality was associated more with the anesthesia than with post-operative complications. Wounds and instruments were washed with 1/1000 Zephiran Chloride. Instruments were washed between implantations.

Implants of testis, ovary and ovotestis were removed from hosts at 14, 30, 60, 90, 120, 150, 180 days and compared with normal controls and with the left gonad of other male chicks of comparable age which had also been treated at 96 hr. incubation with diethylstilbestrol. Histological examination was made on Bouin-fixed tissue cut at 10μ and stained with hematoxylin and eosin. Note was also made of the reproductive status of the host.

The recovery data for implants have been grouped according to type of tissue implanted. Table 1, sections A, B and C, has been arranged to indicate the number of implants which had been made for recovery at each age, the number of implants actually recovered, condition of cortical elements (presence of follicles and/or oögonia in cords or nests), medullary elements (immature versus mature tubules producing spermatozoa), a note if lymphoid elements pre-dominated over gonadal components in the implanted tissue, and the number of ovulating hosts among the birds sacrificed. The last column gives the percentage of implants made which contained recognizable medullary and/or cortical elements.

TABLE 1.

Survival of implants to 0–3-day-old female chick hosts

Survival of implants to 0–3-day-old female chick hosts
Survival of implants to 0–3-day-old female chick hosts

Forty-six per cent, of the testis implants, 20 per cent, of the ovarian and 29 per cent, of the ovotestis implants were recovered. The most persistent element of the implants was the medullary tubule in which both germinal and Sertoli elements could be readily identified. The number of follicles which persisted in the ovarian implants was always considerably below the numbers seen in section of host ovary, and even in cortices containing almost normal numbers of follicles the largest follicular diameter was less than 50 per cent, of that of the host ovary. The medullary component of the ovotestes was identified in all thirty-one of the recovered organs but in only one 14-day implant was a cortical remnant identified. Enough males which had been treated at 96 hr. incubation were left intact to permit sacrifice of three to four for each group. The summary of characteristics of their left gonads is found in Table 2. It will be realized that while all ovotestes were evaluated for presence of a continuous cortex by low power observation before they were used for implantation, there is no way of knowing the original extent of cortical development in the left gonad of birds included in Table 2. The most frequently en-countered characteristics of the intact ovotestes were lacunae and cords of ‘fat-laden’ cells (Nonidez, 1924). These also appear in the normal ovary as modifications of the medullary tubules. Portions of the surface of the ovotestes were often simple cuboidal epithelium, comparable to that of the ovary rather than a fibrous capsule such as is seen in the testes. In some of the ovotestes there were two further modifications which were not common to either the testis or ovary. The first was the development of a rather broad intermediate or transition zone of loose connective tissue located between the cortical oogonial nests and whatever normal medullary components remained. This transition zone, seen so distinctly in ovotestes of 1-day chicks, persists long after the dis-appearance of cortical gonia. The second modification was the appearance of sterile cords or primitive tubules immediately beneath the surface of the gonad and generally close to the intermediate zone if one was present. These structures have been previously described (Wolff & Hampe, 1950) as being the result of a secondary proliferation of sex cords in the ovotestes following the initial effect of estrogenic stimulation of typical cortical cords. The cords referred to appear to be similar to those which formed in remnants of partially extirpated ovaries and in hypertrophied right rudiments of castrate females (Domm, 1927). Regression of the intact ovotestes paralleled that described by others (Wolff, 1935; vanTienhoven, 1957; Haye, 1959). The most persistent gross modification noted was the flattening and wrinkling of the involved testes. Normal medullary tubules with germ cells were present in all specimens examined.

TABLE 2.

Characteristics of intact ovotestes

Characteristics of intact ovotestes
Characteristics of intact ovotestes

It is apparent that the attempts to prolong survival of cortical portions of the ovotestes were unsuccessful. Since the cortex of control ovarian implants also failed to survive, there was no evidence to indicate whether the potential of the oogonia of the ovotestis is less than that of natural oögonia.

The homograft reaction, as evidenced by lymphoid involvement (Tables 1 & 2), was apparently responsible for loss of the majority of implants since the tissues were positioned to render accidental dislodging unlikely. Seven additional ovarian implants not included in the tables, were made to hormonally bursectomized female hosts prior to learning that cell fines derived from the thymus, not the bursa, are apparently responsible for immunological responses to tissue grafts in the chick. At 30 days four of the seven implants were re-covered. All showed some lymphoid involvement, three contained follicles, and one of the three closely resembled the host ovary in number of follicles but these were not equal in size to those of the host. The results were similar to those obtained with untreated hosts.

Cock (1962) reported that ovarian implants made to fully castrated males of various ages retained both endocrine and reproductive capacity to a far greater extent than did those implants made to incompletely castrated males. He observed that ‘the presence of testicular tissue, particularly in large amounts during the initial period, prejudices the survival and normal development of the (ovarian) graft’ . This corresponds to our conclusions that the remaining medullary tissue of the ovotestis may be the factor responsible for cortical regression rather than an inherent inadequacy of the cortical gonia or follicles. Parrott (1960) reported that endocrine capabilities are maintained better than reproductive capacities in stored and transplanted mouse ovaries. Beber (1957) who cultured 15– 18-day embryonic rabbit ovaries and testes individually and in combination, found that testes grew better than ovaries and that in combination the ovaries ceased development or developed into sterile intersexes.

Holyoke (1956) implanted embryonic rabbit testes and ovaries to adult rabbit kidney and found ovarian survival depressed in presence of a companion testis although sex of the host did not appear to play a rôle.

The poor survival of cortical elements of the implants might be related to (1) establishment of vascularity, (2) superfluous nature of the implants from an endocrinological point of view of the host, or (3) inherent instability of the cortex—at least in early stages of ovarian growth. Experimental evidence indicates that early vascularization may be a mixed blessing—serving to support normal metabolism of the tissue but affording more immediate access of the tissue to host immunological mechanisms. Parrott (1960) found that ovarian implants to the subcutaneous site were in an immunologically privileged location in comparison to those made orthotopically in the rat. The tendency for autografts and homografts of mammalian ovaries to be deficient in oocytes has been shown by Buyse (1935), Jones & Krohn (1960), and Mussett & Parrott (1961). Greenwood (1924–5) reported that chick embryo ovaries survived transplantation to the chorioallantoic membrane less well than did testes. Taber (1956) reported poor oögonial survival in perirenal implants of embryonic chick ovaries made to bilaterally ovariotomized or caponized hosts. The number of oocytes lost during the first week of orthotopic implantation in rats has been determined to be 65 per cent, (minimal) and tends to be even higher in ovaries taken from progressively older donors (Mussett & Parrott, 1961). It is possible that the embryonic or neonatal ovary may not be sufficiently stable for transplantation and that the shock to homeostatic mechanisms within the implant may result in regressive changes not seen in implants of slightly older ovaries. The marked difference in ability of dissociated embryonic chick testes and ovaries to reaggregate in culture (Abraham, 1960) may be a reflection of such inherent instability.

There are several points to be made based on this report and reports of others. (1) The use of newly hatched chicks as hosts did result in retention of sufficient numbers of implants to demonstrate the far greater viability of medullary than of cortical components of the chick gonad. (2) The chick chromosomal constitution shows the male with ZZ and the female with ZO complement of sex chromosomes (although the cataloguing of chick chromo-somes is not complete). This suggests that implants of testis and ovotestis such as made in our experiment are not faced with the complication of host intolerance to a sex chromosome such as described for homografts of male (XY) mammalian tissue to female hosts (Eichwald & Silmser, 1955; Lustgraaf et al., 1960). The experimental work with mammalian embryos versus chick embryos has shown a distinct difference in in vivo studies of differentiating gonads. Six-to eleven-day left or right gonads (Wolff, 1946–7) and left or right medullary components (Mintz & Wolff, 1954) from female chick embryos will induce cortical development of the differentiating left gonad of the male when the two are approximated in vivo. Heterosexual pairs of rat gonads have been implanted to the subcapsular site of castrated adult hosts (Macintyre, 1956). In such a combination the ovary was inhibited or transformed but the testis was unaffected. Inhibition of the testis occurred only in a small percentage of cases in which 13-day anlage were placed adjacent to 16– 19-day differentiated ovaries (Macintyre et al., 1959). The development of the right rudiment in partially or completely ovariotomized hens has already been referred to. The contrast of endocrine potential of the embryonic ovary in which both cortex and medulla, left and right gonads, feminize versus the aspect of the more mature ovary in which without inhibitory control of the cortex the medulla will undergo some degree of masculinization with regard to both structure and function is most interesting. Domm’ s report (1927) of feather and sex skin changes following ovariotomy and subsequent hypertrophy of the medullary rudiment on the right side with return of feathers to henny-type plumage but persistence of masculine comb and wattles underscores the dual endocrine rôle of the medullary tissue in the female.

Further investigation is being made to determine the survival potential of implants of cortical material alone. At the present time we are studying the stability of heterotopic autografts of ovarian cortex made at varying ages post-hatch. If such grafts can be made successfully, heterotopic homografts of cortex from ovaries and ovotestes will be followed by attempts at orthotopic cortical implants to hosts in which greater efforts will be made to develop homograft tolerance.

  1. Implants of 1– 3 day chick ovary, testis and ovotestis were made to intact female hosts of the same age to determine the effect of the host’ s endocrine activity on survival of normal ovarian cortex, testicular medulla and the cortex of feminized testes (ovotestes).

  2. Isolation of the ovotestis from an apparently unmodified right testis and relocation to the endocrine environment of the female organism did not enhance persistence of normal follicular development of ovotestes. Cortical development of transplanted ovaries, however, was also severely inhibited.

  3. Medullary tubules were the most persistent elements in implanted tissues.

  4. Spermatogenesis was complete in seminiferous tubules of a number of ovotestis and testis implants recovered from both ovulating and non-ovulating hosts.

  5. The significance of proximity of the cortex of the ovotestis to persisting medullary components, the homograft reaction, and the possible instability of embryonic and neonatal ovaries is discussed.

  6. The medullary components of testis and ovotestis implants survived with a much greater frequency than did portions of the implanted ovaries.

Homogreffes de jeunes testicules, ovaires et ovotestis chez la poule intacte

  1. On a greffé des ovaires, des testicules et des ovotestis de poulet de 1 à 3 jours sur des hôtes femelles du même âge pour déterminer l’ action de l’ activité endocrine de l’ hôte sur la survie du cortex ovarien normal, de la médulla testiculaire et du cortex de testicules féminisés (ovotestis).

  2. L’ isolement de l’ ovotestis d’ un testicule droit apparemment non modifié, et son transfert dans le milieu endocrinien de l’ organisme femelle, n’ ont pas accru la persistance du développement normal des follicules des ovotestis. Néanmoins, le développement cortical des ovaires transplantés a été, également, fortement inhibé.

  3. Les tubules médullaires ont été les éléments les plus persistants dans les tissus implantés.

  4. La spermatogenèse s’ est accomplie dans les tubules séminifères d’ un certain nombre d’ ovotestis et de testicules repris sur les hôtes où ils avaient été implantés, que ceux-ci soient ou non en période d’ ovulation.

  5. On discute la signification de la proximité du cortex de l’ ovotestis sur la persistance des constituants médullaires, la réaction d’ homogreffe, et l’ instabilité possible des ovaires embryonnaires et néonataux.

  6. Les constituants médullaires des implants de testicules et d’ ovotestis ont survécu beaucoup plus fréquemment que ne l’ ont fait les portions d’ ovaires implantés.

We wish to acknowledge the assistance of Raymond Alie whose technical skill contributed so much to the completion of this project. The work was aided by a grant from the National Foundation.

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