After Aristotle's Generation of Animals, there was a scarcity of comprehensive books dealing with reproduction. Lesser works were published by Galen and William Harvey (discoverer of the circulation). A few excellent texts finally surfaced - most noteworthy are Francis Marshall's Physiology of Reproduction, first published in 1910, and Sex and Internal Secretions, which first appeared in 1932. The period spanning the mid-1920s to 30s is arguably the most productive in the history of reproductive research.

Endocrine doctrine. Before 1849 it was widely assumed by the scientific community that integrative activities of the body were the exclusive principality of nerves. Evidence that involvement of the nervous system was only part of the communicative story was introduced by Professor Berthold of Göttingen. Berthold caponized cockerels and observed atrophy of the combs and a decline in sexual behavior. Libido and secondary sex characteristics were promptly restored when testicular tissue was grafted to the intestines (replacement therapy) of castrate birds. Berthold concluded that nerve regeneration could not have been responsible for the rapid reversal of such remote effects. It appeared that a blood-borne substance (hormone) was released from testicular transplants. The experimental approach of "remove-and-replace" would become a cornerstone of endocrine research.

A note of digression: some forty years after Berthold's report of androgenizing effects of the testis, an elderly physician, Charles-Edouard Brown-Séquard, claimed to have rejuvenated himself by injection with aqueous extracts of animal testes; unfortunately, this sensationalized declaration was not authenticated.

Isolation of hormones. The "hormone theory" was not given serious attention until the turn into the twentieth century (Bayliss and Starling, 1904); once accepted, the arduous task then at hand was to isolate and purify the hormones. Structural resolutions of the sex steroid hormones came to fruition in a timely fashion. Crude bioactive extracts of gonadal, pituitary, and placental protein hormones were prepared during the 1920s and 30s. Purification and structural analyses of the protein hormones would prove more difficult than that of the simplistic steroid molecules - amino acid sequencings and determinations of locations of covalent bonds (primary structure) were not undertaken until after World War II. An additional class of hormone-like compounds, the prostaglandins, were discovered during the 1930s.

Oxytocin was the first of the reproductive hormones to be isolated from its organ of storage. In 1906 Sir Henry Dale mentioned that an extract of ox pituitary gland caused contraction of the feline uterus. An additional property of the posterior lobe, increased milk flow in lactating goats, was reported a few years later. By the end of the 1920s glandular preparations of oxytocin (pitocin) had reached the marketplace for induction of labor.

The "roaring 20s" were the pennant years of steroid biochemistry. Estradiol and progesterone were isolated in the United States from porcine follicular fluid and corpora lutea by Allen and Doisy (1923) and Corner and Allen (1929), respectively. Testosterone was identified by Koch and his associates in 1928. Simultaneous feats were accomplished in the European laboratories of Butenandt, Marrian, and Laqueur. Soon the placenta was enlisted with the gonads (and adrenal cortex) as an organ of steroidogenesis.

That the ovaries undergo cyclic changes in response to a circulatory impetus was proposed by Walter Heape in 1905; he suggested that the ovaries were nourished by a "generative ferment" - the provisional forebearer of the pituitary gonadotropins. There was initial conflict over whether the mammalian anterior pituitary gland produced a single gonad-stimulating hormone (as in lower vertebrates) or two different gonadotropins - one that stimulated follicle growth (follicle-stimulating hormone, FSH), and one that caused ovulation and CL formation (luteinizing hormone, LH). Researchers at the University of Wisconsin, especially Fevold, championed the concept of duality. By the late 1930s it seemed certain there were separate follicle-stimulating and luteinizing hormones of pituitary origin. An LH-like pituitary hormone isolated in males stimulated Leydig cells to secrete testosterone, and was called interstitial cell-stimulating hormone (ICSH); notwithstanding, the original female acronyms, FSH (follitropin) and LH (lutropin), remain the favored terms of usage in male physiology.

Prolactin (PRL) was actually the first of the anterior pituitary hormones to be isolated. Stricker and Grueter in 1928-29 described a pituitary factor that stimulated lactation in pseudopregnant rabbits and multiparous dogs, pigs, and cows. Prolactin was further classified as a gonadotropin after it had been shown to stimulate luteal function in some rodents. Forthwith it became evident that prolactin was extremely versatile in its actions among vertebrates - from teleost fishes upward on the evolutionary scale. Effects of prolactin were demonstrated on maternal behaviors, growth, integument, osmoregulation, and energy metabolism.

In 1926 Hisaw reported that serum from pregnant rabbits or guinea pigs induced relaxation of pelvic ligaments in virgin guinea pigs. The relaxant compound, relaxin, was isolated the following year from pig corpora lutea and rabbit placenta. Interest in relaxin waned until the early 1950s, when it was shown to inhibit uterine contractions and enhance cervical softening.

Urine was a plentiful source to look for other water-soluble hormones. Human chorionic gonadotropin (hCG) was discovered in urine of pregnant women by Aschheim and Zondek in 1927. Physiological attributes of hCG were similar to pituitary LH. Gonadotropic activity was also detected in urine of postmenopausal women (human menopausal gonadotropin, hMG). In 1932 estrogen sulfate esters were extracted from pregnant mare urine; since the early 1940s commercial preparations (premarin) have been the most commonly prescribed estrogen replacement for steroid-deficient (eg., postmenopausal) women.

Cole and Hart (1930) isolated a gonadotropin from serum of gestating horses with follicle-stimulating activity - pregnant mare's serum gonadotropin (PMSG). It was assumed that PMSG was of pituitary origin. Later, the endometrium (lining of the uterus) was a suspected source of PMSG. Eventually the origin of PMSG was traced to cells of the chorionic girdle that invade the endometrium ("endometrial cups") - alias equine choriogonadotropin (eCG). An endocrine role for the placenta ascribed earlier by Halban (1905) was securely established.

The prevailing contention was that the gonads produced only lipid-soluble hormones. In 1932 Perry McCullagh found that the testis also contained an aqueous principle that inhibited pituitary hypertrophy in castrate male rats; he named this compound inhibin. An analogous (FSH) inhibitory factor was subsequently recovered from ovarian follicles.

In 1930 two Columbia University gynecologists, Raphael Kurzok and Charles Lieb, observed that semen contained a unique (fatty acid) substance that had a marked stimulatory effect on uterine contractility; these findings were corroborated by Goldblatt in England and Ulf von Euler in Sweden. Von Euler (who would eventually be awarded the Noble Prize for his pioneering studies of catecholamines) named the seminal factor "prostaglandins" - he presumed that these compounds were derived mainly from the prostate gland (actually, seminal vesicles produce much higher amounts). The field of prostaglandin research was then abandoned for almost three decades.

During the 1940s and 50s attention was shifted to embryonic endocrinology. The ground-breaking experiments of Alfred Jost indicated that differentiations of mammalian genital organs were controlled by the presence (male) or absence (female) of testicular hormones - testosterone maintained Wolffian ducts and (a novel protein) Mullerian-inhibiting hormone (MIH) caused regression of Mullerian ducts in male embryos (without these hormones, the female phenotype was expressed).

Proof that the pineal gland was an organ of internal secretion came late. Melatonin, the active ingredient of the pineal gland, was isolated in 1958; it was named as such because of its relationship to both melanin (a pigment of the skin and hair) and serotonin precursor.

Ovarian-uterine/vaginal dynamics. It was known from ancient times that breeding cycles and fertility in animals depended on the ovaries. Much later it became apparent that the ovaries in turn controlled fluctuations in uterine and vaginal activities.

The roles of the ovaries in uterine hyperplasia and pregnancy maintenance were introduced by Knauer in 1900 and Fraenkel in 1903. Uterine growth-promoting effects of the ovaries were attributed to the follicular component. The progestational product was localized to the CL by Ancel and Bouin in 1909. Conversely, at least in some species, the uterus regulated luteal function - Leo Loeb in 1923 found that luteal activity was prolonged in hysterectomized guinea pigs. Ovulation suppression by the CL was inferred by Loeb after he had observed (1909) that ovulation occurred sooner in animals when CL were removed. Antiovulatory effects of luteal extracts were defined by Haberlandt (1919-1923).

On contraceptive grounds there was interest in defining the timing of ovulation with phase of the menstrual cycle. A common fallacy evolved from an analogy made between proestrous uterine bleeding in dogs and menses in women. It was inferred that because dogs ovulated about the time of proestrus, then women ovulate during menses - this created a procreational paradox in some cultures that regarded menstruating women spoiled and unfit for intercourse ("the curse"). Carl Hartman, a well-known comparative reproductive biologist, was instrumental during the 1930s in educating the public. Hartman established that ovulation occurred between the onset of menstrual periods; this information formed the basis underlying the rhythm method of contraception (midway abstinence).

Correlations of ovarian dynamics with vaginal cytology in guinea pigs were first reported by Stockard and Papanicolaou in 1917. In their classic description of the rat estrous cycle, Long and Evans (1922) related temporal changes in ovarian functions to morphology of epithelial cell types. Exfoliated vaginal cells were swabbed onto a glass slide and examined under a light microscope. An outstanding feature of the "vaginal smear" was the presence of cornified (keratinized) cells when animals were in estrus ("heat"). Indeed, the vaginal smear method is still commonly used to monitor stages of the estrous cycle in laboratory rodents. Papanicolaou adapted a swabbing technique ("Pap" smear) for the diagnosis of lower genital tract cancers in women.

Male reproduction. An understanding of the compartmentalized functions of the testis emerged during the first half of the twentieth century. Light microscopic studies of serial cross-sections of testicular tissues clearly indicated that the exocrine (spermatogenic) capacity of the testis was confined to its avascular seminiferous compartment. Bouin and Ancel in 1903 assigned an endocrine role to the interstitial cells described previously by Franz von Leydig. By 1930, the consensus was that Leydig cells, via male hormone, nourished the spermatic tubules. Greep and co-workers in 1936 provided evidence for dual gonadotropic control of testicular functions - Leydig cells were acted upon by LH, Sertoli cells by FSH; albeit refined, this elegant proposal has survived to modern times. Hormonal control mechanisms of mammalian spermatogenesis have since been studied - while androgens and gonadotropins are undoubtedly involved, the business of exactly how and to what degree remains in contention.

That spermatogenesis in many warm-blooded mammals required a temperature lower than core body was suggested by Crew in 1922 and substantiated by Walton (1930). Rates of spermatogenesis were defined during the mid-1900s by Asdell, Ortavant, Clermont, and others. As early as 1897 it was known that testicular spermatozoa were immotile and needed to pass through the epididymis to gain fertility. That sperm cells undergo a final process of maturation within the female tract (capacitation) was recognized independently by Austin and Chang in the early 1950s.

Since the work of an Italian priest, Lazaro Spallanzani, there has been an interest in the preservation of sperm cells for assisted reproduction; he reported (1782) the birth of three pups to a bitch bred by artificial insemination (AI). The benefit of AI for the genetic improvement of livestock was put into practice after World War I by the Russian scientists Ivanoff and Milovanov. The first AI cooperatives were organized in Denmark in 1936 and in the United States in 1938. In 1940 Phillips and Lardy described the use of an egg-yolk diluent to preserve cooled bovine sperm. Antibiotics were added to the sperm preservative recipe by Almquist in 1948. A major boost to the AI industry was the observation by Polge et al. (1949) that inclusion of glycerol in semen extenders protected sperm cells from freeze-thaw damage. The first calf produced using frozen semen was reported in 1952. Frozen spermatozoa were used for human AI by 1953.

The comparative ultrastructure of sperm cells was delineated with the development of transmission electron microscopy techniques; Donald Fawcett, histologist extraordinaire, was a leader in this field.

In 1786 Sir John Hunter, a prominent experimental surgeon, described the atrophy of male accessory glands following castration. Approximately 150 years elapsed before testosterone was shown to restore glandular function in orchidectomized males. The classical work of Huggins during the 1940s, using the canine as an experimental model, demonstrated a dependency of prostatic overgrowth on testicular androgens; androgen withdrawal/antagonism has been a clinical tactic to manage human diseases of the prostate gland ever since.

Detailed chemical analyses of accessory organ secretions (seminal plasma) were carried out during the middle decades of the past century. Seminal fluids contain an array of compounds that have been painstakingly cataloged by investigators like Mann and Williams-Ashman; what purposes many of these substances fulfill, if any, is not yet known.

The emphasis of explorations in reproduction has been, and still is, on the female.

Hierarchy of control. By 1912 the pieces of the puzzle of reproductive control began to fall into place. An Austrian neurosurgeon Aschner found that hypophysectomy (surgical removal of the pituitary gland) by the buccal approach (through the cheek) resulted in gonadal involution. Philip Smith mastered parapharyngeal (through the roof-of-the-mouth) hypophysectomy in the rat. By 1927 Smith demonstrated conclusively that pituitary hormones regulated the gonads. In 1929 Fee and Parkes reported that an intact pituitary gland was required for reflex ovulation in rabbits. At long last the pituitary gland was getting its deserved recognition.

Within a few years it became evident that the anterior pituitary gland was, by reciprocal action, held in restraint by gonadal hormones. The notion of end-organ negative feedback was first introduced by Carl Moore and Dorothy Price in 1932 - the pituitary gland was the inferred target. In 1937 Walter Hohlweg discovered that a single, large dose of estradiol induced ovulation (positive feedback) in juvenile rats. Hohlweg, however, believed that the hypothalamus was the center for steroid feedback. Moreover, experiments by Hinsey, Markee, Everett, and Sawyer indicated that neural stimulation of the anterior pituitary gland was not direct, but routed through the hypothalamus.

A network of portal vessels linking the hypothalamus to the anterior pituitary gland was described by Popa and Fielding in 1930; downward blood flow was demonstrated in the toad by Houssay in 1935, and confirmed in mammals the following year (Wislocki and King). The portal system allowed the hypothalamus to regulate the anterior lobe as authenticated by Green and Harris in 1947. During the early 1960s McCann demonstrated gonadotropin-releasing hormone (GnRH) activities in rat hypothalami. The hypothalamus had replaced the anterior pituitary gland at the height of the "hierarchy of control" (Figure 1-2).

How secretion of prolactin is controlled by the hypothalamus was confounded by observations (~ 1960) that hypothalamic extracts contained both prolactin-releasing (PRF) and inhibiting factors (PIF). The inhibitory influence of the hypothalamus over prolactin prevailed - removal of the anterior pituitary gland from its neurovascular connections (eg., by transplantation beneath the kidney capsule, pituitary stalk-section, or hypothalamic ablation) favored secretion. There is still some dissension over the chemical character of PIF and PRF ... "factors" are elevated to the status of "hormone" once a consensus is reached.

Bioassay. With identification of hormones came an interest in methods of routine detection - a logical approach was to test a sample for its ability to cause a biological response (bioassay) (unaware of the hormone in particular, recall that Berthold put this principle to work). Refinements in bioassay methodology allowed hormones to be quantified from an arithmetic scale constructed from the results of graded reactions. An increase in ovarian weight was used to estimate FSH-like activity of pituitary extracts or plasma. Luteinizing hormone stimulated ovarian ascorbate depletion (the physiological significance of this relationship remains uncertain). An increase in weight of crop sacs (a storage pouch for regurgitated food located just beyond the esophagus) of pigeons was used to measure prolactin. Gross and microscopic alterations in the reproductive tract were used to monitor steroid hormones. Many bioassays (eg., contraction of smooth muscle) lacked specificity.

It was sometimes necessary to measure hormones by indirect means. Measurement of increased gonadotropin production was taken to imply an increase in secretion of GnRH. Diminished plasma FSH ascertained by lack of ovarian responsiveness was an indication of elevated inhibin.

Some bioassays were designed to yield only qualitative (yes-or-no) results. Rabbits (or mice or frogs) were injected with human urine (hCG) and their ovaries examined for ovulation points to detect pregnancy (Friedman test).

Most bioassays are now mainly of historic interest and have been succeeded in research and clinical laboratories by more practical and sensitive methodologies (some are, however, still used to establish comparative potencies of different lots of standard hormonal preparations). Classical bioassays for the reproductive hormones are listed in Table 1-2.

Genetics of sex determination. Very few topics in biology have been given more conjectural attention than what causes a developing individual to become either male or female. The basic rule of heredity - that each parent contributes one hereditary unit for each phenotypic trait expressed by the offspring, was delineated in 1865 by the Austrian monk Gregor Mendel (he worked with crossbred garden peas). The haploid nature of meiotic gametes, and the restoration of the diploid condition by their union, was recognized in 1883 by van Beneden.

By 1901 it was evident that a sex (from the Latin word sexus, which means division) difference existed in the chromosomes. Experiments using the Drosophila fruit fly indicated that a particular chromosome (X) was responsible for sex determination. The presence of two X chromosomes was female-determining (1X = male). The Canadian cytogeneticist, Murray Barr, recognized that one of the two X chromosomes in somatic cells becomes randomly inactivated and condensed (forming a Barr body). Among mammals (as in Drosophila, but in contrast to birds) the male was heterogametic (XY).