Feline embryo ~ 21-23 days

Key words: Embryo, feline, organogenesis, crown-rump, genital ridge.

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Click on the image to enlarge it.

A feline embryo estimated to be approximately 21 to 23 days of age based on its (difficult to ascertain) crown-rump length and physical appearance in an image from Knospe, C. 2002.

Besides the intriguing organogenesis, of special interest to the author was the genital ridge. It is particularly obvious in this specimen. This structure is well known to reproductive biologists as the progenitor of the gonads; a ridge populated by primordial germ cells from the yolk sac. These cells  migrate to the ventral aspect of the mesonephros on either side of the spinal column. In this specimen it is not possible to determine if the gonads will develop into ovaries or testes.

The author extends thanks to Dr Glenda Wright (gwright@upei.ca) and Dr  Vanmathy Kasimanickam (vkasiman@wsu.edu) for their editorial comments on this image.

Selected references:

Davidson, A.P. et al. 1986. Pregnancy diagnosis with ultrasound in the domestic cat. Vet. Radiology and Ultrasound. 27:109-114

Knospe, C. 2002 Periods and Stages of the Prenatal Development of the Domestic Cat. Anat. Histol. Embryol. 31:37–51

Luvoni, G.C. 2013. Ultrasonographic Foetal Biometry in Dogs and Cats. WSAVA2013 - VIN

Nelson, N.S. and Cooper, J. 1975. The growing conceptus of the domestic cat. Growth 39:435-451

Topie, E. et al. 2015. Early pregnancy diagnosis and monitoring in the queen using ultrasonography with a 12.5 MHz probe. J.Feline Med.Surg. 17:87-93

Persistent Müllerian duct syndrome

Keywords: feline, cat, cryptorchid, testes, Müllerian, inhibin, AMH

A feral tomcat was admitted for routine castration. The right testis was present in the scrotum and the cat had a penis with well developed barbs, suggesting the presence of androgens. The left testis was was not palpable in either the scrotum or inguinal canal. Therefore an exploratory laparotomy was performed to locate and remove it.  A uterus, normal in appearance was present in the abdomen. At the cranial tip of the left uterine horn, a structure resembling a small testis (testicle) was present. The right uterine horn passed through the right inguinal canal. The scrotal testis on that side was removed and the tip of the right uterine horn drawn cranially into the abdominal cavity so that a hysterectomy could be performed. The uterus with both gonads is shown below.

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Dr Heather Hillier (hhillier@stjohns.ca) brought this case to the attention of the author and retains copyright for the image shown above.

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The image above shows the histology of the retained gonad (top) and the scrotal testicle (bottom). The retained gonad was a testis with poorly developed seminiferous tubules containing cells that were a mixture of spermatogonia and sertoli cells. Some tubules contained only sertoli cells. Areas between the tubules were populated by leydig cells and a cell type that the author was not able to identify with certainty. These were labeled "Unknown cell type". The nuclei of these cells bore a strong resemblance to those of the leydig cells but their vacuolated, and small amount of cytoplasm was not typical for normal leydig cells. Perhaps they were indeed leydig cells but a sub-population showing functional artifacts.

The lower part of the image shows the seminiferous epithelium of the descended testicle. It was normal in appearance; spermiation was even occurring in some areas. 

The image below shows a complete absence of spermatozoa in a cross section of the epididymis from the retained testis (the top inset). In terms of the non-functional seminiferous tubules in that testis, this was expected. It was surprising however, to find no spermatozoa in the epididymis of the scrotal testis. This may indicate that there was non-union between that testicle and its epididymis.

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A cross section of the uterus revealed the expected tissue layers of a normal uterus. However, the endometrial glands were poorly developed. Nevertheless, many glands contained eosinophilic material, suggesting secretory activity.

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Discussion
This cat showed an unusual form of intersex known as persistent Müllerian duct syndrome (PMDS). It would not be surprising to find PMDS in any mammal but it is well known in humans and has also been described in dogs, notably Miniature Schnauzers. A remarkable case with a completely dissected tract was described by Haibel, G.K. and Rojko (1990) in a goat. A presumptive diagnosis of PMDS has been made in a male cat with pyometra but to the author's knowledge, this is first case where the condition has been verified in a cat histologically.

Some basic endocrinology will help in understanding this unusual condition.

AMH and inhibin are closely related dimer proteins, part of a large family of growth factors that control protein secretion as well as cell growth and differentiation. Importantly and especially in the case of AMH,  they also control programmed cell death (apoptosis). Both AMH and inhibin are produced by sertoli cells in males and the granulosa cells of follicles in females. The principal function of inhibin is to decrease FSH secretion in both males and females. That of AMH, is to prevent FSH binding to pre-antral follicles and (of great importance in this case) to cause apoptosis of the Müllerian system in males.

Recall that the Müllerian system persists by default in embryos i.e. if the the gonads (male or female) are removed from an embryo, the Müllerian system will persist and grow into a cranial vagina, cervix and uterus. However, if an embryo has testes, it is the presence of sertoli cells in those testes, producing AMH, that cause cell death and regression in the Müllerian system. Of course, the presence of testes also causes masculinization of the embryo. Returning to the case in hand, this animal had two testicles; one scrotal and the other retained; both were likely sources of AMH.

Strictly speaking, an animal with PMDS is a male pseudohermaphrodite because its genital tract does not match the sex of its gonads. But unlike other male pseudohermaphrodites, its external genitalia are normal in appearance. It is the internal genital tract that does do not match the sex of the gonads.

Disentangling oneself from all definitions, it is perhaps easiest for the reader to picture a male with PMDS as being normal in every sense (masculine appearance, libido and even fertility in a few cases) but for the retention of a uterus that would otherwise have regressed under the influence of AMH. In a few cases, a normal Wolffian system exists, abaxial to the uterus, explaining how some men with PMDS are fertile.

In dogs, it has been shown that AMH is actually present in many cases of PMDS but probably because of receptor site abnormalities, it is unable to exert its effect on the Müllerian system. In humans, that is also true in some cases but in others, the concentrations of AMH are low or undetectable. In a small proportion of cases reported in humans, both AMH concentrations and receptor sites were normal. In those cases, the cause of the condition was unclear. It is not yet known if all these situations occur in dogs as well.

Most cases of PMDS are associated with autosomal recessive abnormalities. The basic karyotype of affected males is usually normal i.e. 46XY in the case of men, 60XY in the goat mentioned earlier and 78XY in the case of dogs. Karyotyping was not performed on the cat described here.

PMDS is usually discovered at surgery and affected males are often cryptorchids; unilaterally or bilaterally (the goat was not cryptorchid). Scrotal hernias containing the testis and part of the uterus are also part of the PMDS syndrome. As expected, the cryptorchid testes in these cases occasionally become neoplastic as well.

Important: As seen in the canine section of LORI, it is possible that this cat could have been an XX pseudohermaphrodite (a case of "sex reversal") because XX pseudohermaphrodites may also show persistence of the Mullerian system in the face of anti-Mullerian hormone production. However, to date, no cases of XX sex reversal have been described in cats. Therefore the cat in this entry was in all probability, an XY male pseudohermaphrodite and therefore, a case of PMDS.

Selected references:

Belville, C. et al 1999. Persistence of mullerian derivatives in males. Am. J. Med. Genet.89:218–223.

Berkman, F. 1997. Persistent mullerian duct syndrome with or without transverse testicular ectopia and testis tumours. British J. Urology. 79:122-126

Hagjer, S et al. 2015 Intra-abdominal seminomas in bilateral undescended testes in a patient with persistent mullerian duct syndrome. Hellenic J. Surgery 87: 493-496

Haibel, G.K. and Rojko, J.L. 1990. Persistent Miillerian Duct Syndrome in a Goat. Vet Pathology. 27:135-137

Kaore A et al 2012. Persistent Mullerian Duct Syndrome NJIRM.3: 153-154

Knebelmann, B. et al 1991. Anti-Mullerian hormone Bruxelles: A nonsense mutation associated
with the persistent Mullerian duct syndrome. Proc. Natl. Acad. Sci. 88:3767-3771

Meyers-Wallen, V.N. et al 1989. Müllerian inhibiting substance is present in testes of dogs with persistent müllerian duct syndrome. Biol. Reprod. 41:881-888

Meyers-Wallen, V.N. et al 1993. Mullerian inhibiting substance is present in embryonic testes of dogs with persistent mullerian duct syndrome. Biol. Reprod. 48:11410-1418

Nayak, V.J. et al 2014. Persistent mullerian duct syndrome: A case report and review of the literature. Int J Appl Basic Med Res. 4:125–127

Schulman, J. and Levine, S.H. 1989. Pyometra involving uterus masculinus in a cat
J Am Vet Med Assoc.194: 690–691

Weissbach, L. et al. 1999, Prognostic factors in seminomas with special respect to hCG: results of a preospective multicentric study. Eur Urol. 36:601-608

Meyers-Wallen, V.N. 2009. Review and Update: Genomic and Molecular Advances in Sex Determination and Differentiation in Small Animals. Reprod.Dom.Anim. 44:40–46

The yolk sac and zonary placenatation in late gestation

Keywords: yolk, sac, feline. exocelom, 55 days

Interestingly, the yolk sac persists for most of pregnancy in carnivora; it is shown here in a feline fetus 55 days old. In other domestic species, it is no longer visible by the end of the first third of pregnancy.

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Note: We have adopted the term chorioallantois (allantochorion) with ease, understanding that it is a combination of two membranes i.e. the chorion and allantois. Yet, in advanced gestation, we persist in referring to an"amnion" when in reality it is the amnioallantois; a combination of the amnion and allantois. The author is of course playing the devil's advocate here, knowing full well that these statements will never achieve traction in wider biology....

To understand this image and the presence of an overt exocelom in the carnivora, one should consult the diagram in another entry in LORI. The exocelom is present in all embryos and early fetus in all species but is rapidly ablated in those animals except for dogs and cats. It is easily appreciated during cesarian sections in companion animals, occasionally mystifying the surgeon  

The urachus is present but not visible in this image. It runs of course in the umbilical cord existing into the allantoic cavity which is no longer present; stripped away here to show the amnion.

The specimen from which this small section arises in shown below. An in-depth discussion of the image is superfluous here because a similar LORI entry is to be found elsewhere. The image does however provide an alternative view of the same anatomy.

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Extreme cystic endometrial hyperplasia

Keywords: feline, uterus, CEH, hyperplasia

The uterus of an adult domestic long haired cat. 

Image size: 1000 x 1333px. Author and owner of copyright: Dr Stephanie N. Simpson (stephanienicolesimpson@gmail.com)

The cat was presented in good health with a mildly distended abdomen. When its uterus was exteriorized, this appeared to be a case of pyometra. However after hysterectomy and upon opening the uterus, no pus was present. Instead, the enlargement of the uterus had been caused by the remarkably distended cystic structures shown here. Because of financial constraints, the histology of the uterus was not examined. In the absence of histopathology, this was presumed to be an extreme expression of cystic endometrial hyperplasia (CEH). 

The case is remarkable because of the dramatic nature of the CEH.

CEH in cats has been eloquently examined, summarized and referenced by Dr Robert Foster (rfoster@uoguelph.ca) and readers are encouraged to visit his site for more information. 

Essentially, CEH is associated with age and occurs in approximately 20 to 40% of cats where no clinical signs are present. The endocrinology of CEH has been investigated and is thought to be linked to a higher-than-normal expression of estrogen receptors in the endometrium. It frequently occurs in a luteal phase but as for pyometra in this species, not all cases occur after ovulation. In dogs by contrast, elevated progesterone, CEH and pyometra are virtually inseparable.

Twenty one to 22 day feline embryos

Keywords: anatomy, embryology, feline, embryo, exocelom, yolk sac, allantois, amnion

A feline pregnancy, 21 to 22 day into gestation. In this case, the tract was removed from a feral cat so the actual duration of gestation was not known. The length of gestation was verified by comparing the development of these embryos with embryos of known gestation in Knospe's publication of 2002 (see reference).

The tense, spherical shapes of an early feline pregnancies are evident in this image. These lend themselves to easy identification during transabdominal palpation in cats. The spherical shapes become less distinct after 35 days of gestation. In this pregnancy, two of the embryonic-placental units have been opened. It was of course necessary to transect the chorioallantois and release the allantoic fluid to expose the embryos within their amnions.

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The embryo at left has been partially removed from the uterus. The soft tissue indicated by the black arrow is the inner chorioallantoic surface of the zonary band of the placenta. The blue arrow indicates the same tissue but in that case, it is in situ within the uterine lumen.

The diagram below illustrates the cardinal features of an embryo at this stage of development. Note that the chorioallantois has not yet elongated.  Elongation becomes evident as pregnancy advances. 

A reminder: The outermost membrane in all embryos is the chorion. The allantoic sac lies inside the chorion. The fluid-filled allantoic sac covers the amnion either partially or completely depending on the species. Among the domestic species, coverage over the amnion is only complete in horses. Where the allantois lies adjacent to the chorion, it gradually fuses with that membrane. That fusion results in the formation of the chorioallantois or allantochorion (either term is acceptable). The amnionic membrane also fuses with the allantoic membrane, forming the amnioallantois. Except in texts on embryology, amnioallantois is a term seldom seen in the literature; instead it is commonly referred to as the amnion.  The exocelom is a substantial cavity, lying between the yolk sac and the allantois and in an early embryo such as this, between the amnion and allantois as well (see the green color code in the images). The presence of the exocelom is often neglected although it persists and is obvious as a space in the umbilical cord even at the time of parturition in carnivores. It is not seen in advanced gestation in farm animals.

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The image below shows an embryo taken from the top image. To help the viewer understand the structures that are visible here, a key has been created directly below this image.

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The image below is a key for the image above. Recall that the allantochorion has been removed, so a double line (the membrane is a two-part structure) was added by the author to show its approximate position before the embryo was exposed. Also note that the zonary band of the placenta has been turned back to expose the embryo. If the zonary band was to be replaced, returned as shown by the yellow arrow, it would cover all the structures seen here.

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The paper by Knospe, C. 2002 included under references, contains numerous referenced benchmarks in organ development in feline embryos at 21 to 23 days of age . Paraphrasing some of those benchmarks:

The eyelids have started to form and the external ear has been established. The outlines of the future digits appear as rays. The genital protuberances have formed and  the skeleton and muscles have begun to differentiate. The tongue and the primitive larynx are formed and the thymus, thyroid glands and heart appear. Pleural cavities are developing around the lungs and the lungs already show bronchi. The stomach is visible and the liver is developing. Of particular interest here is the fact that the indifferent gonads have developed but both the mullerian and wolffian ducts are still present.

The image below shows another embryo viewed from its anti-mesometrial aspect. In the carnivores, the yolk sac lies adjacent to the mesometrium. The embryo and its membranes lie against the backdrop of the placental attachment zone. Because the allantochorion had to be transected to reveal this view, the allantois and its fluid have been lost. Three fluid filled cavities remain. The halo of the amnion lies closest to the embryo. Below that is the elongated yolk sac. With a circumference larger than that of the yolk sac is the clear outline of the exocelom.

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Reference:

Knospe, C. 2002. Periods and Stages of the Prenatal Development of the Domestic Cat Anat. Histol. Embryol. 31, 37-51

Feline placentation

Keywords: feline, placenta, anatomy

Feline gestation is approximately 64 days, starting about a day after breeding (cats are induced ovulators). Therefore, feline gestation is marginally longer than canine gestation.

Using body, crown-rump and head diameters from various published sources, the pregnancy below was estimated to be approximately 53 days of age. The first image serves as a key for the main image. Unfortunately, the manner in which the fetuses were arranged for photography suggests that they were somehow attached to one another. They were in fact, three separate fetuses.

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In these specimens, the allantochorionic membranes of these fetuses were transected and everted (turned inside out), releasing the allantoic fluid from each fetus and exposing their internal chorionic surfaces (in reality, their allantochorionic surfaces because the allantois and chorion fuse as gestation progresses). The amnionic fluid remains within the amnions of the fetuses.

The gross anatomy of feline placentation is described as "zonary" and is described in greater detail elsewhere in LORI.

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Note the pellets of meconium; probably an indication of fetal stress. In advanced gestation, it is not uncommon to find yellowish amniotic fluid which in turn, has stained the fetus a yellowish color. Meconium is often found in the lungs of those fetuses as well.

Meconium staining is generally associated with fetal stress but a literature search will reveal that the pathophysiology is not at all clear. Fetuses with meconium staining are not always in state of acidosis and the overriding sympathetic tone associated with stress does not explain anal relaxation and increased peristalsis.

As shown above, the extra amnionic umbilical cord is not well defined in cats. The same is true of dogs as illustrated elsewhere in LORI. One could argue that these species do not even have extraamnionic cords. This is because the exocelom spreads out widely where the base of the exocelom and yolk sac remnant attaches to the placental zonary bands.

As mentioned elsewhere, the feline placenta does not have a green hematophagus border as is seen in canids. However, the mechanism for iron absorption appears to be similar in both species

Conjoined feline twins

Keywords: feline, conjoined, fetus, dystocia, radiograph

This image is provide courtesy of Dr Ruth Massey (masseyruthellen@gmail.com, holder of copyright). It shows two conjoined feline fetuses and a radiograph of that specimen. Although remarkable, it is far from unique in the subject of fetal malformations. In fact, conjoined fetuses are quite common in humans; about 10 in every million births. Conjoined fetuses have been seen in all of the domestic species. Indeed, one of the references given here shows that it has been formally documented in cats as well. In birds, especially ducks, conjoined hatchlings are reported with some frequency; the same is true of fish.

In the case of the cat reported by Camon et al, the degree of fusion and exact levels of anatomical compromise were described accurately. In this case, the specimen was collected during an emergency cesarean section and there was little opportunity to examine it in detail.

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The kittens died either during or shortly after delivery.

Although one of the twins were originally reported as being male and the other female, it should be appreciated that gender determination is often challenging in newborn kittens, even in normal individuals. The ano-genital distance is often used for this determination, that of the male being greater than the female. In a malformed fetus, this measurement may have been probably unreliable. Attention is drawn to this finding because a  case of conjoined fetuses of different genders has never been reliably documented; indeed, it is highly unlikely to occur. This case then, is used as basis for a brief summary on the nomenclature and pathogenesis of conjoined twins.

First, it is common to use the “pagus” (fr. Gk  pagos meaning “fixed”) together with a particular body part to describe where the fetuses are conjoined. In this case for example, both the thorax and heads are joined, therefore this specimen is a cranio-thoracopagus conjoined fetus. Interestingly, if there are two heads (often facing in the opposite direction) the specimen is referred as a “Janisceps” fetus. This term is more correctly spelled “Janusceps” because it refers to the Roman god Janus, who had two faces, one facing towards the future and the other backward to the past.

Most readers will be familiar with the fact that the incidence of fraternal (non-identical) twins is related to the incidence of multiple ovulations, not split embryos. Spontaneous multiple ovulations are well known to have a heritable basis but this is not generally the case for identical twins (or triplets), certainly not in mammals. In rare cases of inbreeding  (in Burmese cats for example) it may have a genetic basis but generally speaking, it is not known why an early would split partially or completely into two or more identical embryos. However, using teratogens such as vincristine, urethane, nitrofurans, ethanol, thalidomide and DMSO, conjoined twins have been experimentally produced in small mammals.  In fact, even variations in temperature and alterations in embryo culture have been used to induce conjoined twins. In spontaneous cases, the cause is seldom known.

It should be appreciated that conjoined twins only occur when splitting of the embryo occurs at a relatively late stage, i.e. when the primitive streak is forming. This too, is when the early amnion and chorion have already formed. Therefore, conjoined embryos usually have a shared single amnion and a shared single chorion. This situation only accounts for a very small proportion of twin embryos. The majority of twin embryos are formed when the embryo splits earlier than this. In humans for example, it is known that about 30 percent of identical twins are formed in the first few days of life. In that case, two completely separate entities develop, with separate amnions and separate chorions. These are known as dichorionic-diamnionic twins. However, close to 70 percent of identical twins form a little later, when a single chorion has already formed and the inner cell mass then splits into twin embryos, each forming its own amnion. These individuals become monochorionic- diamnionic-, identical twins. As mentioned, only the small remaining portion of identical twins are monochorionic-monoamnionic. If these proportions hold true for domestic animals it may help to explain why conjoined twins are such a rare occurrence.

Interestingly and intuitively, both normal and conjoined monoamnionic-monochorionic identical twins are mirror images of one another; in humans, their finger prints are mirror images of one another. As expected, situs inversus occurs in one of the twins as well. Situs inversus was not reported by Camon et al and was not examined in the case presented here. It would be interesting to investigate this in other cases of conjoined twins seen in domestic animals. In humans, it is also known that conjoined twins are significantly more common in female than male twins but this has not been documented in other animals, perhaps because of the rarity of the condition.

References:

M. H. Kaufman M. H. 2004The embryology of conjoined twins Childs Nerv Syst 20:508–525
Camon, J. et al 1992. Morphology of a dicephalic cat. Anat Embryol 185:45-55