In the study of sexual development, two life stages may be considered of prime concern: the period of prenatal or neonatal sexual differentiation and puberty. The former stage is considered crucial for the organization of behavioral and physiological reproductive patterns seen in the adult. The latter is crucial for the activation of these patterns or the bringing of these patterns to expression. This is as true for humans as it is for other species (Diamond, 1965; 1968). In the female, recent studies of sexual differentiation have concentrated on the malleability of the neural tissues associated with mating behavior and the hypothalamic-pituitary-gonadal axis in reproduction (e.g., Phoenix, Goy, Gerall, and Young, 1959; Barraclough and Gorski, 1962), while the studies of puberty seem to concentrate on the initiation of ovulation and the ability to produce young (e.g., Greenwald and Peppler, 1968) or probe the general question of adolescent sterility (Hartman, 1931). Some experimental studies have attempted to induce puberty (e.g., Ramirez and Sawyer, 1965) and from them extrapolate mechanisms of the neural-endocrine influence on puberty and reproduction. Aside from the singular work of Blandau and Money (1943) in the rat, few studies are known, however, which have simultaneously investigated the integration of sex behavior processes and reproductive abilities along the spectrum of parameters during pubertal development. This is so despite the fact that successful reproduction is dependent upon the proper coordination of sexual behavior as well as other physiological parameters.
The present investigation is an attempt to more fully and completely analyze pubertal maturation in a most rapidly developing species and to allow a more complete and comprehensive comparison between components of the sexual behavior-reproductive system of the female during development. Furthermore, an attempt is made to more closely correlate the pubertal findings of sexual behavior with other developmental parameters, particularly in regard to the question of adolescent sterility. For this work a rapidly maturing laboratory species, the golden hamster, was chosen.
This study investigated the spontaneous and simultaneous development of reproductive features including first appearance of estrus, ovulation, vaginal opening, vaginal cyclicity, fertilizability of ova, and ability to bear and deliver viable young.
MATERIALS AND METHODS
Female golden hamsters bred in our own laboratory and left with littermates until weaning were used. Twenty-two animals from seven litters were weaned at Day 17 or 18 and kept in groups of three or four. They were housed in opaque, white plastic cages, 10 x 12 x 6½ inches until temporary removal for behavioral tests or separation in breeding cages. All animals were weighed prior to each behavioral test. The light schedule was adjusted to provide a 6:00 P.M. to 6:00 A.M. dark cycle.
On the day of birth (equals Day 0) and again on Days 5, 10, and then daily, 14 of the 22 intact females were examined until the first appearance of a patent vaginal orifice was seen. Starting at 17 days of age all females were weighed and examined daily for the first display of behavioral and vaginal estrus, ovulation, and the ability to conceive, bear, and parturate. Behavioral tests for estrus were initially conducted between 9:00 PM and 11:00 PM and consisted of placing the female to be tested into an observation cage with an experienced adult male for 5 min. Observations were conducted under red light. Behavioral receptivity was quantitatively evaluated by recording the number of 15-sec intervals (quartiles) during which the female exhibited lordosis in response to the investigating-stimulating male. A maximum score would thus be 20 (4 quartiles x 5 min). Females manifesting vaginal estrus without showing a lordosis response on their first test or if showing 10 or less response quartiles were retested between 11:00 PM and 1:00 AM the same evening.
All females showing vaginal estrus, and all those exhibiting lordosis were left overnight with males and checked for vaginal sperm the next morning.
Any female exhibiting lordosis during the test period was laparotomized the next morning and the left ovary and oviduct removed and examined for evidence of ovulation. In case of doubt the ovaries were prepared for histological inspection. The oviducts were flushed with 0.9% NaCl solution in search of ova. When fertilized eggs (in the pronuclear stage) were found in the oviduct, the female was separated in an individual cage and observed for a least 18 additional days (normal adult gestation = 16 days). For those females not bearing or parturating young from this first pairing, mating was again initiated at the next available estrus and these animals were again followed for their ability to bear and parturate. Any female exhibiting the sequential vaginal secretory conditions typical of estrus followed the next morning by a postovulatory condition were laparotomized and inspected for tubal ova and the presence of corpora lutea even if estrous behavior was not displayed.
Vaginal estrus and cyclicity was determined by utilizing the categories of vaginal secretion enumerated by Orsini (1961). Essentially, a clear, translucent and stringy mucous secretion is associated with a preovulatory condition (our late Day 4) ; an opaque, white, thick, odiferous mucoid mass is associated with a postovulatory condition (our Day 1) ; and an opaque, waxy, yellowish plug is associated with diestrus (Day 2). Vaginal secretions are usually absent on the second diestrus day (Day 3) and early on Day 4.
Throughout the experiment all animals were kept in air-conditioned quarters at a temperature of 68-750 F. Diet consisted of standard rat/hamster chow supplemented with dog chow and water ad libitum.
The most obvious result is that full reproductive maturity and integration of all the various components involved in successful pregnancy may be said to occur by the age of 35 days in the female golden hamster. The various facets, however, appear to develop in a related but independent manner. All results are listed in Table 1.
|TABLE 1 - REPRODUCTIVE DEVELOPMENT IN THE GOLDEN HAMSTER|
|Litter||Animal||Age (days)||Weight||Lordosis intensitya||Littering||Remarks|
|Vaginal discharge||1st Estrus|
|1st Observed estrus||1st Clear stringy||1st Post-ovulatory||At 1st observed estrus (g )||9 PM-11 PM||11 PM-1AM||1st Preg||2nd Preg|
|10||2 silent||28||32||―||―||Double hemicastrate|
|12||1 silent; 35||31||32||70||18||Double hemicastrate|
|13||1 silent; 36||32||33||70||0||19||Double hemicastrate|
|6||18||1 silent; 32||28||29||66||2||6||Double hemicastrate|
a Number of 15-sec intervals (quartiles) during which the female exhibited lordosis in response to the investigating male ; a maximum possible score is 20.
Apparently, the earliest reproductive feature to develop is vaginal patency. Between 5 and 10 days of age, 11 of the 14 female hamsters inspected showed vaginal patency, all 14 vaginae were opened by 15 days of age.
The first estrus occurred in one female at 26 days of age. Twelve additional females came into first heat between 28 and 35 days of age. Typically, this first behavioral heat was preceded by a clear mucous vaginal estrous condition followed by ovulation and fertilization. For two females, estrus was neither preceded by vaginal mucous typical of estrus nor followed by ovulation. One female (L3-10) did not come into heat during the extended observation period ; while she did show cyclic vaginal changes and ovulation.
While first estrus occurred among the animals over a span of 17 days (26-43 days), within each litter the span was much narrower, usually 3-5 days, and never more than 6.
During the first estrus of every female observed, the typical firm lordosis stance was seen. The intensity of this first spontaneous estrus, however, as measured by duration of lordosis display was generally weaker in these young animals than in animals older than 35 days. Approximately 25% of younger females did not respond to the stimulating males at the initial 9:00 PM test, but did so at the subsequent testing between 11:00 PM and 1:00 AM. Those females first responding after 35 days of age did so on the initial 9-11 PM test.
When the young female is not sexually receptive, in contrast to the typical aggressive pattern and fighting displayed by an adult female, the adolescent tries to avoid contact, climb the wall of the test enclosure, rolls on her back or assumes a standing “boxing” pose to parry the mounts of the male. No direct aggressive contact resulting in any bodily damage to either the male or female was seen.
Vaginal secretions typical of the estrous cycle were consistently the earliest precursor of behavioral estrus ; usually a clear stringy mucoid secretion but occasionally of milky color. The first vaginal secretion in several females was waxy.
Ovulation, as determined by laparotomy after vaginal estrus and a postovulatory vaginal secretion was not always preceded by estrus. Five females from three litters displayed anestrous ovulations (“silent heat”); one female did this twice on subsequent cycles. Strong lordosis behavior was seen during the subsequent cycles of the remaining four females. These females were sacrificed after the two hemicastrations needed for the determination of ovulation.
Ovulation was not evidenced by tubal ova or ovarian corpora on Day 27 in the female displaying the earliest estrus or in another female on Day 34. The first ovulation was detected on Day 29. This was not preceded by vaginal signs of estrus although a strong behavioral response (18 quartiles) was seen. Starting at 32 days of age all females typically ovulated after vaginal and behavioral estrus, and fertilization resulted from all but one mating.
Despite the normal appearance of fertilized ova in the mated females, on initial conception only three of nine that occurred before the female was 34 days of age terminated successfully, while after 35 days of age three of five delivered young. Subsequent pairings resulted in fruitful pregnancy and parturition in all cases. In these pregnancies gestation length was between 15 and 16 days. Litters from the younger mothers were usually smaller than from older mothers. All mothers regardless of age successfully nursed and cared for their young. No cannibalism was seen (as indeed is rare in our colony).
The relationship between weight and day of first estrus was computed. No significant correlation was seen (r = .35).
In contrast to the rat, in which vaginal patency usually develops about a week prior to the first estrus and may even follow it (Blandau and Money, 1943), in the hamster the vagina opens quite soon after birth independently and without relation to ovulation or estrus. Our findings of early opening correspond to those of Kupperman, Greenblatt, and Hair (1944), Bond (1945), Brunner and Witschi (1946), and Ortiz (1947), but differ from reports of late opening (Day 27-35) given by Sheehan and Brunner (1945). Reasons for the apparent wide disparity are not clear.
Normal estrus in 26- and 28-day-old females is perhaps the earliest known occurrence of spontaneous sexual receptivity reported for any mammal. This, coupled with our findings that the remaining females will mate by 43 days of age is comparable to the findings of Selle (1945), Greenwald and Peppler (1968), and Peczenik (1942). Reed and Reed (1946), however, report the first mating in their colony occurred at 46 days of age. It may be noteworthy to consider that the stimuli surrounding determination of estrus varied considerably among these studies. Selle (1945) observed his females with seven males for an hour each night while in the Greenwald and Peppler study ( 1968) the females and males were permanently caged together and mating determined by subsequent sperm checks. In our test situation, the female was exposed to a male only for the 5-min observation periods during the active nocturnal hours.
The findings that females within the same litter tend to come into first estrus and ovulate at ages more like their sibs than other age mates and findings of anestral ovulations concentrating in the same litters are provocative. It may indicate a genetic factor need be considered in pubertal development.
In general, the initial estrus follows appropriate vaginal changes and precedes ovulation. Our findings of anovulatory estrus and its counterpart anestrous ovulation, however, are similar to findings seen in the developing rat (Blandau and Money, 1943) in which ovulation and estrus may independently manifest themselves. Also in agreement are our findings in regard to the onset of estrus in which more mature females come into estrus earlier in the evening then do less mature ones. Harvey, Yanagimachi, and Chang (1961) reported 100% of 100 mature female hamsters displayed estrus by 11:00 PM; 23% of our 22 did not show heat until after 11:00 PM on their first estrus. In the hamster the intensity of the lordosis response is less strong on this first heat than subsequently. This has been reported also in the rat (Blandau and Money, 1943).
Few animals mating prior to 34 days of age were able to complete a successful pregnancy. Selle (1943) and Bond (1945) report copulations as early as 30 days of age but first littering not occurring until much later. Deanesly ( 1938) found the earliest pregnancies successful at 50-70 days of age. According to our observations, the ova produced on these initial ovulations (Day 29 to Day 44) appeared to have been successfully and normally fertilized. The lack of successful subsequent pregnancy indicates that some other hypothalamic (uterine?) processes remain to develop. The hemispayed young hamster seems no less capable of bearing and parturating than the intact adult female. The number of pups born to our young females and those from Selle’s intact females are comparable to these reported born to 1- and 2-month-old females by Soderwall et al. (1960). The gestation length of all our first-term pregnancies was between 15 and 16 days. This too is in keeping with the findings of Soderwall et al. that until 5 months of age the average gestation length is less than 16 full days. The similarities indicate that hemicastration did not seem to significantly affect the number of young born and the absolute number of functioning corpora lutea, if at least four are present, is not crucial for successful gestation. It may be significant that resorption is not uncommon in the hamster and usually occurs after Day 9 of gestation (Purdy and Hillemann, 1950). With our smaller number of initial embryos resorption might have been minimal.
The weight of the hamster at first estrus or first ovulation does not correlate with the age of these events. From the present study in concert with data ranging from rat (Long and Evans, 1922; Blandau and Money, 1943) to human (Donovan and van der Werff ten Bosch, 1965) we suggest puberty be considered as not dependent upon any single facet of reproduction and sexual maturity, but rather reflect the many processes involved. Thus we would define puberty, for females of any species, as that period during which adult-like sexual behavior and physiological reproductive processes become activated and normally become coordinated. Organization of these reproductive parameters may be said to occur over a relatively short period. Coordinated activation occurs over a more protracted duration. Component or coordinated activation may never occur ; adult females sterile for one reason or another may mate but never become pregnant or may ovulate and never mate. They, having passed through the period during which puberty normally occurs, would be considered reproductively postpubertal and “mature.”
It is obvious there remains much to be elucidated regarding those factors which actually begin to coordinate the adult sex behavior-reproductive system. Studies recently completed indicate that the neural tissues mediating sexual behavior activation are not the limiting factors. We can induce, with estrogen and progesterone, estrus in castrated and intact females as young as 19 days of age and in all cases by 28 days of age (Diamond and Yanagimachi, 1970). This indicates that the proper activation of coordinated mating and reproductive activities probably depends upon those crucial hypothalamic-pituitary-ovarian processes organized pre- or neonatally.
Appreciation for assistance in the execution of this research is extended to Mr. Alfonso Liacuna and Mrs. E. Barbara Zegart. This research was supported in part by Public Health Service Grants HD 03394, and HD 02066, of the National Institute of Child Health and Human Development of the National Institutes of Health, and in part by funds from the Ford Foundation. The senior author is supported in part by a Lederle Medical Faculty Award.