Sexual Differentiation and the Testis

An AMH-null male mouse can produce functional sperm and retain a uterus. How can those outcomes coexist? You can answer by separating testis determination from the signals a testis sends. Then you can use the same distinction between a signal’s source and its target to understand adult sperm production.

One gonad, two possible fates

A bipotential gonad is an embryonic gonad capable of developing into a testis or an ovary. In most mammals, including humans, the Y-linked gene SRY initiates testis determination by increasing SOX9, a transcription factor that changes gene expression in supporting-cell precursors. Those precursors become Sertoli cells, the supporting cells of the testis.

In the mouse model, positive feedback helps maintain SOX9 expression and reinforce Sertoli differentiation. The opposing ovarian network includes WNT4, RSPO1 and beta-catenin, with FOXL2 contributing to ovarian identity; ovarian development is an active genetic program. Sertoli cells also shape the signals governing developing Leydig cells and germ cells. A gonad’s fate and its effects on other organs are separate causal steps.

For SRY’s identification by Sinclair and colleagues in 1990 and Koopman’s 1991 mouse work, see sex linkage and beyond.

An enhancer is regulatory DNA that increases a gene’s expression in a particular cellular context. Croft and colleagues’ 2018 human SOX9 work implicates enhancer duplication or deletion in altered testis determination, extending the mechanism beyond coding sequences. Their reporter assays tested cloned regulatory fragments in transfected cells; such activity does not establish a whole-gonad expression threshold. Croft’s study

Two ducts, two hormones

Early mammalian embryos have both Wolffian, or mesonephric, ducts and Müllerian, or paramesonephric, ducts. In the standard human developmental map, testosterone from Leydig cells, the steroid-producing testicular cells, maintains Wolffian derivatives: epididymis, vas deferens and seminal vesicles. The epididymis is the duct where sperm mature after leaving the testis; the vas deferens carries them onward.

Fetal Sertoli cells secrete anti-Müllerian hormone (AMH), which causes Müllerian regression. Without testicular hormones, the reproductive tract develops along the female-typical pathway: Müllerian derivatives include oviducts, uterus and upper vagina. This describes tract development; it does not make ovarian differentiation passive.

Alfred Jost removed gonads from rabbit fetuses in utero, while they remained in the uterus, and inspected their later anatomy. If both tract patterns required a hormone from their corresponding gonad, removing either gonad should prevent its tract pattern. Instead, early testis removal permitted Müllerian development and loss of most Wolffian structures; removing fetal ovaries did not prevent the female-typical tract pattern.

Timing mattered. Jost’s 1947 report describes males castrated on fetal day 20 that retained female genital tracts yet still had rudimentary prostatic buds; later operations preserved more established male structures. His results support a requirement for fetal testicular influence during particular developmental intervals in rabbits, rather than continuing hormone dependence of every structure. Jost’s report

These experiments ruled against a required ovarian secretion for rabbit female-tract development. Gonad removal alone could not tell the testicular signals apart. Jost’s later experiments did: testosterone given in place of a testis maintained Wolffian ducts but did not remove Müllerian ducts, so he inferred a separate Müllerian inhibitor. Molecular work later identified that inhibitor as AMH.

Testosterone for the ducts, DHT for the outside

In humans, 5-alpha-reductase type 2, encoded by SRD5A2, converts testosterone to dihydrotestosterone (DHT). Human fetal DHT signalling supports masculinization of the external genitalia and prostate development. Testosterone and DHT act through the same androgen receptor (AR), a hormone-responsive transcriptional regulator; see nuclear receptors.

In the human developmental map, the genital tubercle contributes to the glans penis or clitoris, and the labioscrotal swellings become scrotum or labia majora. With androgen signalling, urethral-fold fusion contributes to the penile urethra. Common embryonic starting structures therefore need not yield the same final anatomy.

Read this table as predictions when one component is missing from the start of development, before the relevant signal would act, with the other pathways intact. The species labels matter, particularly for the human SRD5A2 row.

Missing component and system	Müllerian outcome	Wolffian outcome	External outcome
Testis: early rabbit gonadectomy	Retained	Largely lost	Female-typical
AMH: male mouse null mutant	Uterus and oviducts retained	Male derivatives retained	Male-typical
AR: XY Tfm mouse	Regressed	Not maintained	Female-typical
SRD5A2: human mechanism	Usually absent	Generally retained	Reduced masculinization

Human SRD5A2 deficiency is inherited as an autosomal recessive trait, so affected people carry disease-causing variants in both copies of SRD5A2, usually one from each parent. Some variants abolish enzyme activity; others reduce it. In 46,XY humans (one X and one Y chromosome), complete AR loss likewise separates testis formation and usually intact AMH action from absent androgen response. These are distinct molecular mechanisms.

The X-linked Tfm mouse, named for the historical mutation “testicular feminization,” tests receptor failure; Lyon and Hawkes’s paper established its X linkage. Loss of AR-mediated feedback predicts increased LH drive, but testosterone concentrations depend on species and testis function; normal or high testosterone is a human AR-loss pattern, not a guaranteed Tfm-mouse value.

In 1994, Behringer, Finegold and Cate generated mice lacking MIS, another name for AMH, by targeted gene disruption. If AMH is the separable inhibitor, these males should retain Müllerian organs despite having testes: they did, and could produce functional sperm. The experiment establishes AMH’s necessity for Müllerian regression in mice; it does not locate AMH’s receptor or measure a human response curve. The AMH-null study

Try predicting each anatomical panel before changing a signal in the “Build the tract” model below; its enzyme toggle represents the human SRD5A2 mechanism.

Switch each part on or off

A testis forms (SRY raises SOX9, giving Sertoli cells) Off: the table’s rabbit gonadectomy row AMH is made and acts Off: the male mouse null mutant The androgen receptor works Off: the XY Tfm mouse 5-alpha-reductase type 2 works Off: the human SRD5A2 mechanism

In this sketch: an XY embryo with a bipotential gonad; every switch is a complete loss from the start of development with the others intact, as in the lesson’s table; the switches combine findings from rabbit, mouse and human, so two or more switches off at once is a prediction from these rules, not an observation; if the receptor is off the external genitalia are drawn female-typical whether or not the enzyme works; “reference” is a made-up baseline for LH and testosterone, with no numbers, concentrations or units; the LH and feedback arrows come from the lesson’s general statements, not from fetal data; the external-genitalia names (glans, scrotum, labia) are human structures.

A testis sends two separable signals: AMH removes the Müllerian ducts, and testosterone acting through the androgen receptor maintains the Wolffian ducts; in humans, conversion to DHT by 5-alpha-reductase type 2 also supports masculinization of the external genitalia.

Two compartments in the adult testis

The seminiferous tubules are tubes containing developing germ cells; the interstitium is the tissue between them. Interstitial Leydig cells make testosterone under the pituitary hormone luteinizing hormone (LH). LH-receptor signalling increases cyclic AMP and cholesterol delivery into mitochondria; the shared steroid machinery is covered in adrenal glands and the cascade in cAMP and PKA.

The other pituitary gonadotropin, or gonad-stimulating hormone, is follicle-stimulating hormone (FSH); it acts on Sertoli-cell FSH receptors, and both gonadotropin receptors use G-protein signalling. Sertoli cells nourish germ cells and respond to local testosterone. Intratesticular testosterone is far higher than circulating testosterone, so blood concentration alone cannot specify local androgen support.

The blood-testis barrier is a junctional seal between neighbouring Sertoli cells, rather than capillary endothelium. It separates a basal compartment, containing spermatogonia (mitotic germ-cell precursors) and early spermatocytes (cells entering meiosis), from an adluminal compartment, toward the lumen, containing later meiotic cells and spermatids (haploid products of meiosis). Mouse evidence shows junction disassembly and reassembly as spermatocytes cross; epithelia explains the junction structure.

Testosterone feeds back on the hypothalamic-pituitary axis, restraining LH and FSH output, partly through conversion to estradiol; Sertoli-derived inhibin B is conventionally assigned selective inhibition of FSH. Apply the loop logic from endocrine axes; the ovarian axis is the next lesson.

In Coviello and colleagues’ three-week study of healthy men in 2005, testosterone injections given with a placebo suppressed LH and FSH, and intratesticular testosterone fell by about 94%. Men who also received low-dose hCG, which acts on the LH receptor, kept intratesticular testosterone in the normal range. Circulating and local androgen compartments can therefore move differently.

Larose and colleagues’ 2020 SCARKO mice lacked AR specifically in Sertoli cells: surviving spermatocytes could synapse and recombine chromosomes, yet failed to acquire competence for meiotic division. Cytology and single-cell RNA sequencing showed that mutant germ cells stayed in the gene-expression state of early prophase (leptotene and zygotene), a transcriptional block, supporting a Sertoli-mediated requirement without implying that every early meiotic event requires androgen or that humans arrest identically.

A conveyor belt with a fixed rhythm

Spermatogenesis is sperm production through germ-cell proliferation and differentiation. Spermatogonia divide mitotically; a spermatocyte’s two meiotic divisions yield four haploid products; spermiogenesis reshapes spermatids into spermatozoa; spermiation releases them from the seminiferous epithelium. Heller and Clermont’s timing work and the cell sequence are in animal reproduction; the chromosome steps are in meiosis.

An epithelial cycle is the interval until the same combination of germ-cell stages reappears at one location in a seminiferous tubule. In humans, it is conventionally about 16 days, while the textbook total from a differentiating spermatogonial lineage to spermiation is about 74 days. Heller and Clermont’s original 1963 labelling study estimated about 64 days for the whole process. Their 1964 analysis gave about 74 days once spermatogonial divisions were counted, and Amann’s 2008 review recommends 74. Some textbooks still give 64, which leaves those divisions out, so always check which starting cell a number uses. Like a conveyor with several batches on it, the epithelium repeats its local pattern before any one lineage completes the whole journey.

Misell and colleagues in 2006 labelled DNA with deuterated water in 11 men with normal sperm concentrations, who drank it daily for three weeks while semen was sampled every two weeks for up to 90 days. Label appeared in ejaculated sperm after 64 ± 8 days, reported as mean and standard deviation, with a 42–76-day range. This measured label-to-ejaculate lag includes post-testicular transit and storage; its start and endpoint differ from the textbook total.

Worked problem: count cycles, then add transit

Use the conventional human values above. Assume steady production and a constant cycle length within each individual.

Lineage cycles = 74 days / (16 days per cycle) = 4.625 cycles, about 4.6 cycles.

Check by multiplication: 4.625 cycles x 16 days per cycle = 74 days. A single epithelial cycle therefore does not represent the entire lineage’s development.

For the tracer calculation, make an explicit simplification: label incorporation occurs only during the last premeiotic S phase, the DNA-replication phase in preleptotene spermatocytes. Because the water was taken for weeks, real label availability can mark other dividing precursors, such as spermatogonia, too. In this made-up example, assume 3.5 cycles remain to spermiation and epididymal transit takes 10 days, with no additional storage delay; neither assumption is a measured result from Misell’s study.

Remaining testicular interval = 3.5 cycles x 16 days per cycle = 56 days.

Predicted label-to-ejaculate lag = 56 days + 10 days = 66 days.

The measured mean plus or minus one standard deviation spans 64 − 8 = 56 days to 64 + 8 = 72 days. Your 66-day prediction falls inside that interval. Agreement does not validate the assumed 3.5 cycles or the transit time: different component delays could give the same total. The observed range shows variation in the measured lag, rather than an exact universal sperm-production clock. Because semen was sampled only every two weeks, part of that spread also reflects the sampling interval.

In scrotal mammals, the testis is cooler than the core, with countercurrent heat exchange between incoming arterial blood and the surrounding pampiniform plexus, a network of testicular veins.

Erection and ejaculation: the reflexes

Erection is filling and stiffening of erectile tissue as inflow increases and venous drainage is restricted. Pelvic parasympathetic pathways include nitrergic nerves, which signal with nitric oxide (NO); endothelium also supplies NO. NO activates guanylyl cyclase, increasing cyclic GMP (cGMP). Protein kinase G links cGMP to lower cytosolic calcium and smooth-muscle relaxation; PDE5 breaks down cGMP, as covered in cGMP signalling.

Cavernosal smooth muscle surrounds blood-filled spaces called sinusoids. Relaxation of this muscle and arterial smooth muscle increases filling, which compresses veins beneath the tunica, the surrounding fibrous covering. Connect this local response with smooth-muscle mechanics and autonomic outflow.

Rajfer and colleagues’ 1992 experiments linked electrically evoked relaxation of isolated human cavernosal strips to endogenous NO synthesis. These surgically obtained tissue strips test local signalling, rather than the integrated neural initiation of erection in healthy volunteers.

Emission moves sperm and glandular secretions into the urethra through predominantly sympathetic control of the vas deferens and accessory glands, with bladder-neck closure. Expulsion is the rhythmic outward movement driven by somatic pudendal motor output to muscles including bulbospongiosus. Ejaculation combines these coordinated events; its motor output differs from the relaxation that permits erection.

Design problem

Mice lacking Sertoli-cell AR from fetal life make almost no round spermatids and no elongated spermatids. A colleague argues that this reflects failed Sertoli maturation rather than an ongoing adult requirement. Design an experiment that separates those explanations.

One way to do it

Delete AR only after Sertoli maturation in adult mice carrying a conditional AR allele. This allele has receptor DNA between recognition sites for the enzyme Cre. Target Cre to Sertoli cells to remove that DNA; verify AR loss in Sertoli cells and retention in Leydig and peritubular cells. Include conditional-allele mice without Cre, Cre delivery to wild-type testes, and a contralateral control, with matched handling.

Collect a time series scaled to mouse development: about 8.6 days per epithelial cycle and roughly 35 days for spermatogenesis, allowing cells already beyond a block and stored sperm to clear. Score stage-specific meiotic markers and epididymal sperm; test barrier integrity with a tracer. Measure serum LH and testosterone: a relatively unchanged Leydig compartment is a prediction to test, rather than an assumption guaranteed by Sertoli specificity.

If adult signalling is required, predict meiotic arrest and eventual depletion of epididymal sperm. Willems and colleagues reported adult Sertoli AR deletion with meiotic arrest and no mature epididymal sperm at 40 days, supporting that prediction. Their adult-deletion experiment

Quantify mosaic deletion and control for delivery injury. Persistent sperm after incomplete deletion would not establish receptor independence, and one arrest phenotype cannot identify the responsible androgen-regulated genes. The inference remains specific to adult mouse Sertoli function.